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Cancer cytogenetics
5th year
seminar
RNDr Z.Polívková
Cancers = heterogenous diseases – initiation
and progression are promoted by
aberrant function of genes, that regulate
DNA repair, genome stability, cell
proliferation, cell death, cell adhesion,
angiogenesis, invasion and metastasis –
so called „ cancer genes“
Cancer is caused by stepwise accumulation of
numerous genetic and epigenetic changes
Driving force of tumorigenesis - genomic instability
= increased tendency to alteration in genome
Overview of the major mechanisms to maintain genomic stability during the cell cycle
Major mechanisms involved in
maintaining the genomic stability
1. Fidelity of DNA replication (S- phase)
2. Accurate segregation of chromosomes (mitosis)
3. Precise repair of DNA damage (throughout cell
cycle))
4. Cell cycle check point
2.Segregation of chromosomes in mitosis:
- Chrom. condensation
- Sister chromatid cohesion
- Kinetochor assembly and attachment
- Centrosome duplication and separation
- Spindle formation
-Chromatid segregation
- Cytokinesis
4.Cell cycle check points:
- G1/S check points
- G2/M check points
- Intra S check points
- Spindle check points
-Post mitotic check points
1. Fidelity of DNA replication:
- DNA polymerase
- Mismatch repair
- Replication licensing
- Maturation of Okazaki fragments
- Restart of stalle replication fork
- Telomere maintenance
- Preservation of epigenetic signatures
According Shen, 2011
3. DNA repair
- DNA damage signalling
- DNA repair pathways:
Excision repair: NER, BER
Repair of double strand breaks: HR, NHEJ
Deregulation of responsible genes leads to genomic instability
Interindividual variability in cancer expression due to:
•
•
differences in the amount of DNA damage
capacity to repair DNA damage
Both influenced by genetic predisposition and by environmental
factors, including life-style.
Individual response to exogenous and endogenous genotoxins
due to genetic polymorphisms:
• of xenobiotic-metabolizing enzymes
• of genes of DNA repair or genes of folate metabolism
= „low penetrant genes“
Approx. 350 genes is connected with tumors
„Cancer genes“:
•
protooncogenes
•
tumor suppressor genes
DNA repair genes - mutator genes
Gene functions can be influenced by:
• gene polymorphisms
• alteration of copy number (amplifications, deletions,
duplications, changes in chromosomal number)
• changes of gene structure, chromosome structure
(translocations, inversions etc.)
• gene mutations (substitution, deletion, insertion in coding
sequences or splicing sites)
• epigenetic modifications (imprinting, DNA methylation and
histone modification – histone acetylation/deacetylation,
methylation or phosphorylation)
Activation of oncogenes (change of protooncogene to
oncogene) through:
• mutation
• structural rearrangement (reciprocal translocation, inversion)
• amplification (double minutes or HSR=homogenously staining regions)
• epigenetic changes
• virus insertion
Inactivation of tumor suppressor genes through:
• mutation
• deletion
• epigenetic modification
• mitotic recombination
Iniciation/promotion theory of tumor origin
Prokarcinogene
Metabolic.activation
Ist phase enzymes
Ultimative
carcinogen
Iniciation
1-2 days
Detoxication
IInd phase enzymes
Promotion
10 years
Progression
 1 year
v
Normal
cell
Iniciated
cell
Preneoplastic
cells
Tumor cells
CHA and tumors
1.
Specific CHA in tumors - CHA is primary event in tumor origin
rearrangement in neighborhood of protooncogenes:
- abnormal activity of product
- abnormal gene expression
rearrangement only in tumor cells (chronic myelogenous leukemia, Burkitt
lymphoma)
- deletion of tumor suppressor genes
in tumor cells or constitutional aberrations (heterozygosity)
(e.g. retinoblastoma)
2. Heritable syndromes with increased chromosome breakage
defect of reparation or replication  high risk of malignancies
Chromosomal study in tumors:
Role: - diagnosis and subclassification of haematologic malignancies
- rational selection of therapy, targeted therapies
- prognostic informations
- monitoring of treatment effect, residual leukemia ..
- study of mechanism of carcinogenesis
CHA in tumors:
balanced without loss or gain of material :
translocations, inversions
unbalanced: with loss of material: deletions, monosomies
with gain of material: duplications, trisomies, polyploidies,
amplifications
Primary changes connected with initiation of malignant process
Secondary changes – connected with progression of disease, with
genome instability
Chromosomal aberrations as primary changes connected
with initiation of malignancy
Translocations – 2 types of translocations
1. Translocations leading to fused genes (genes with function in cell
division regulation or differentiation)
Ph1 chromosome in chronic myelogenous leukemia (CML)
= reciprocal translocation 46,XX or XY,t(9;22)(q34;q11)
protooncogen abl transfered from 9q to 22q near the gene bcr 
fused gene bcr/abl  abnormal product = chimeric protein
with constitutively active tyrosin kinase activity –
breaks in introns of genes
Ph1 in CML good prognosis
during blastic crisis another chromosome changes
In ALL (acute lymphoblastic leukemia) other site of break in bcr gene
Ph1 in ALL = bad prognosis
Cme.medscape.com
Fused gene
brc/abl
Wysis 1996/97
Other examples of fused genes:
ALL
t(1;19)
good prognosis
der(19)t(1;19)
bad prognosis
t(12;21)
good prognosis
acute promyelocyt.leu (M3)
t(15;17)
good prognosis
acute myelocytic leu (M2)
t(8;21)
good prognosis
ALL and AML
t(4;11)
bad prognosis
2. Translocation of protooncogenes to position, where they are
abnormally stimulated to transcription
Burkitt lymphoma (BL) – B lymphocytes
t(8;14)(q24;q32) also in other lymphomas
protooncogen myc transfered from 8q to 14q – next to promotor of
broken gene for heavy chain of immunoglobulin  abnormal
stimulation of gene activity  abnormal amount of normal product
t(8;22) or t (2;8) – next to strong promotor of genes for Ig light chains
T-lympho malignancies - breaks near genes for T-cells receptors
Restricted to cells in which genome undergoes somatic rearrangement (e.g.VDJ
recombination of Ig genes) as a part of process of maturation to effector cells
(B,T lymphocytes)
ncbi.nlm.nih.gov
Translocation produces premalignant clone – probably other genetic
changes (mutations, epigenetic changes..) are necessary for full
malignancy
Translocations (balanced) are relatively frequent cause of
malignancies
Most of translocations or inversions were detected in haematologic
malignancies,
In solid tumors translocations are less frequent (and rearrangements are
more complex)
Fused genes encoding:
factors necessary for haematopoetic differentiation –
chimeric product of fused gene increases aberrant transcription
or represses transcription of genes involved in
differentiation
• transcription
e.g. product of fused gene PML/RARα = t(15/17) = chimeric receptor activates
histon deacetylase complex* → transcription repression of genes for myeloid
differentiation → accumulation of immature myeloid cells in acute promyelocytic
leukemia
• tyrosine kinases (regulators of proliferation) – fused gene product =
chimeric protein – constitutive activity – uncontrolled cellular
proliferation
•Histon acetylation removes positive charge of histones – it decreases interaction with negatively charged DNA
in open (active) chromatin
Histon deacetylation restore positive charge leading to tight interaction DNA with histones and condensation of
chromatin to inactive state - inaccesible to transcription factors
CLINICAL UTILITY OF TRANSLOCATIONS:
Targeted therapy:
e.g. first successfully targeted therapy = Imatinib (Gleevec) = tyrosin kinase
inhibitor - good response to treatment in patients with
bcr/abl fusion (t 9/22)
acute lymphoblastic leukemiae (ALL), acute myeloid leukemia (AML)
-second-generation bcr/abl inhibitors = dasatinib, nilotinib
(in resistancy to Imatinib)
All-trans retinoid acid (ATRA) effective in patients with fusion PML/RARα
(t15/17) in acute promeylocytic leukemia (APL)
ATRA reverse transcriptional repression by disrupting interaction of
PMR/RARα protein with histon deacetylase complex that promotes
transcriptional repression
Origin of fused genes and other chromosomal
rearrangements:
Critical lesions = DSB (double strand breaks) - caused by exogenous
(radiation, chemicals) and endogenous factors (reactive oxygen species..)
DSB also consequences of normal cell processes as V(D)J recombination of
B cells and T cell receptor genes, class switching, meiotic recombination …
missrepair of double strand breaks – aberrant recombination
Specific fusion – influenced by position of chromosomes in interphase and
by the presence of sequentional homology in the sites of breaks
role of „ fragile sites“ (=sites of genome instability)
„ Fragile sites“ and tumors
„Fragile sites“ (FS)
• sites of genome instability on chromosomes
• late replicating
• nonrandom loci – disposed to breaks and exchanges
• manifested as gap or break under condition of replicative stress (i.e.inhibition of
DNA synthesis by aphidicoline, 5.azacytidine, BUdR)
FS – common
- rare in < 5% of population (connected with expansion of triplet repeats,
e.g.FRAXA)
In sites of common FS tumor supressor genes and protooncogenes
are located
Common FS = target site of mutagenes/carcinogenes action, site of
integration of oncogene viruses
52% of all translocations in tumors have sites of breaks in FS (Burrow et al. 2009)
CHA as primary event in initiation of malignancy:
Deletions of tumor suppressor genes
Retinoblastoma (Rb) – eye cancer of children
heritable type (familiar or „de novo“ origin) - AD (with reduced
penetrance)
sporadic type – nonheritable – usually afects only one eye
• heritable Rb – 1st step - germinal mutation or deletion in all cells of body
= heterozygote (constitutional abnormality)
2nd step: somatic mutation in one cell of retina = loss of
heterozygosity (LOH)
loss of heterozygosity by somatic recombination
del(13)(q141-142)
• sporadic Rb – both mutations somatic in one cell of retina
Heterozygosity for mutation or deletion =
predisposition to tumor
Imprinting of tumor suppressor gene =
only single functional copy
Retinoblastoma
Mutation of second
allele in one somatic
cell = loss of
heterozygosity
heterozygote
Heritable
RB/rb or RB/Mutation or
deletion
Sporadic
Loss of function:
mutation, deletion,
loss of whole
chromosome, mitotic
recombination
→
→
→
Mutation of both alleles consecutively in one somatic cell
Knudson´s two-hit hypothesis of loss of function of tumor suppressor gene
Heterozygosity in daughter cells
replication
mitotic
recombination
chromatid
segregation
In mitosis
+
heterozygosity
Loss of heterozygosity by mitotic
recombination
+
Loss of heterozygosity
Interstitial deletion 11p
Wilms tumor = nephroblastoma
WT1 locus on 11p13 mutation or deletion
Wilms tu = isolated or a part of
syndrome - WAGR association
(Wilms, aniridia, urogenital anomaly,
mental retardation)
Deletions of tumor suppressor genes –esp. in
epithelial tumors
Gain of material
Amplification of oncogenes:
•
„double minutes“ = amplified circular oncogenes
(extrachromosomal)
•
HSR (homogenously stainin regions) =
amplification and recombination of oncogenes
tandemly to chromosome
•
or insertion of amplified sequences to different sites on
chromosomes
Amplification especially in solid tumors:
e.g.: N-myc in neuroblastoma, cyclin D1 in many tumors (carcinoma of
oesophagus), cyclin D2 in ovarian and testicular cancers
Amplification (gene overexpression) often includes more
than one gene – probably contribute to tumor phenotype
Targeted therapy: Herceptin (trastuzumab)=monoclonal
antibody targeted to Her2/ERBB2 oncogene (= tyrosinkinase receptor) in women with breast cancer and
amplification of this oncogene
Amplification of some „cancer genes“ is associated with
therapeutic resistance (e.g. amplification DHFR gene connected
with resistance to methotrexate, amplification of bcr/abl gene
in CML patients resistant to imantinib/Gleevec, amplification of
gene for androgenic receptor in prostate cancers resistant to
endocrine therapy)
Imprinting and tumors
Tumors: - inhereted or induced mutations of protooncogenes,
tumor supressor genes
- epigenetic changes = changes in methylation
(imprinting) of these genes
Imprinted protooncogenes – error in imprinting → activation
of imprinted allele (biallelic expression) = oncogenes
Imprinted tumor supressor genes – loss of function of
one allele only (active allele) = loss of gene function
only 1 step needed for loss of function = increased
sensitivity to tumors
Polymorfism of imprinting of some genes in population
e.g.tumor supressor genes WT1(11p13)
IGF2R(6q26)=receptor for IGF2 (used for intracellular degradation of IGF2)
usually biallelic expression, but in some peoples monoallelic (=imprinted)
imprinting of these genes = predisposition to tumors
Methylation = reversible process – possibility of therapy of
tumors caused by aberrant methylation??
Chromosomal changes as a consequences of malignant
process, genomic instability
→ deregulation of genes resposible for segregation of chromosomes or
cytokinesis – changes in chromosome number
Gains of material: intragene duplications, duplications of genes,
groups of genes, chromosomal parts or whole chromosomes
Duplication, trisomy : e.g.+8 in blastic crisis in CML, ANLL, MDS
Hyperdiploidiy, polyploidy – hyperdiploidy in ALL = good prognosis
but hypodiploidy = bad prognosis
Loss of genetic material: deletions inside genes, deletions of whole
genes, groups of genes, chromosomal parts or whole
chromosomes - deletions of tu su genes, loss of noncoding genes
(e.g.micro RNA –role in posttranscriptional regulation of gene
expression)
Loss of genetic material = loss of tumor suppressor genes,
loss of micro RNA encoding genes (miRNA - role in
posttranscriptional regulation of gene expression)
Gain of genetic material = gain of (proto)oncogenes
Oncogene Her-2/neu amplification in breast cancer cells– FISH method
Trisomy and tetrasomy in cells of breast tumor
Chromosome loss in cells of breast tumor
New methods of chromosomal study in tumors:
FISH, comparative genomic hybridization and variants of
these methods (mFISH, m-band, array CGH,..)
Array CGH: comparison of tested and normal DNA, both stained
by different fluorochromes – mixture of both is applied to a slide
with thousands of spots of reference DNA sequences to hybridize
- gains and losses of genetic material are detected by computor
as spots of different colours
Specific aberrations = markers for prediction of
disease outcome or response to treatmennt
Identification of genes for therapy or prevention
!! Complex, multiple changes = bad prognosis,
bad response to treatment !!
Karyotype of breast cancer cell
Chromosome instability syndromes
Common features:
• AR inheritance
• increased sensitivity to UV light (sun light)
• hyper - or hypopigmentation
• small stature
• defects of immunity
• increased sensitivity to radiation, chemical mutagens (breaks,
chromosome exchanges, sister chromatid exchanges)
• increased spontaneous level of chromosome aberrations or
increased level of CHA after induction by mutagenes
• increased risk of malignancy !!!
error in DNA reparation or replication
Fanconi anemia (FA)
panmyelopathy with bone marrow failure leading to pancytopenia
skeletal anomalies (thumb, radius), growth retardation
hyperpigmentation
microcephaly, defect of thumb and radius - in 50% of patients
CHA: breaks and chromatid exchanges (multiradials, komplex changes)
heterogenic: several genes (7genes FANCA-G)
- defect in DNA repair activation
Bloom syndrome (BS)
low birth weight, stunted growth
sun sensitivity of the skin
immunodefect (B-lymphocytes)
facial butterfly-like lesions with telangiectasia
most families of Ashkenazi Jewish origin
CHA: breaks, exchanges between homologs, increased level of sister chromatid
xchanges (SCEs)
defect of replication, DSB reparation (DNA helicase-gene BLM)
Ataxia teleangiectasia (AT - Louis-Bar syndrome)
progressive cerebellar ataxia, growth retardation
sensitivity to radiation
oculocutaneous teleangiectasia
immune deficiency (cell immunity)
„café au lait“ spots on skin
CHA: rearrangement of chromosomes Nos 7, 14 or 2,22
= sites of T-cell receptors genes and Ig heavy chains genes
defect of DNA repair (ATM gene – protein kinase regulates TP53,
signal recognition)
Xeroderma pigmentosum (XP)
erythema after UV irradiation of the skin - atrophy,
teleangiectasias
sensitivity to UV and ionizing radiation
skin cancer
CHA: spontaneous level not increased, increased respons to induction of aberrations (UV)
defect of DNA repair (excision, postreplication repair,
repair of DNA strand breaks ) 7types: genes XPA-G + XPV
Nijmegen breakage sy
growth retardation, mental retardation
microcephaly, atypic facies
immunodefect
CHA: rearrangement of chromosomes Nos 7, 14
Rearrangement→failure to produce fully functional immunogluobulins
and T-cell receptors → immunodeficiency
Error in reparation of DNA double strand breaks
gene NBS1 (nibrin)
Syndromes connected with premature aging
Werner sy
Cataracts, subcutaneous calcification, changes of skin, premature hair greying,
premature arteriosclerosis
defect of exonuclease and helicase activity, gene WRN
Cockayne sy
Stunted growth, mental retardation, deafness, premature senility
defect of DNA excision repair (NER)
• Willman CH.L. and Hromas R.A.: Genomic alterations and chromosomal
aberrations in Human Cancer - Google
• Fröling S.and Dohner H.: Chromosomal abnormalities in Cancer,. N Engl J
Med 2008, 359, 722-34
• Mitelman Database of Chromosome Aberrations and Gene Fusions in
Cancer (2013). Mitelman F, Johansson B and Mertens F (Eds.),
http://cgap.nci.nih.gov/Chromosomes/Mitelman
--• Thompson &Thompson: Genetics in medicine, 7th ed.
Chapter 16: Cancer genetics and genomics: Oncogenes, Tumor- suppressor
genes (including Retinoblastoma,Caretaker genes in autosomal recessive
chromosome instability syndromes, Cytogenetic changes in cancer, Gene
amplification)
Chapter 6: Principles of clinical cytogenetics:Mendelian disorders with
cytogenetic effects, Cytogenetic analysis in cancer
+ informations from presentation