Concept of „Read and Write“ genome and
consequences of its application in oncology
prof. A.Vašků
TBCM, September 22, 2017
22. 9.2017 A11/114 Campus
8:30-10:10
Concept of „Read and Write“ genome and consequences of its application in oncology
(prof. A.Vašků, 2 hrs)
29. 9.2017 A11/114 Campus
8:30-9:45
„Next generation sequencing“ in oncology (doc. Mráz, 1.5 hrs)
6. 10. 2017 A11/114 Campus
8:30-10:10
Importance of cytogenetics in hematological malignancies (doc. Ševčíková, 1.5 hrs)
13.10. 2017 A11/114 Campus
8:30-10:10
Importance of epigenetics in oncology (doc. Slabý; 1.5 hr)
20. 10. 2017 A11/114 Campus
8:30-9:45
Pathogenesis of leukemia´s (prof. Mayer, 0,5 hod)
Pathogenesis of multiple myeloma (doc. Ševčíková, 1 hr )
27.10. 2017 A11/114 Campus
8:30-9:20
Pathogenesis of solid tumors (doc. Křen, 1 hr, přednáška)
Theoretical Bases of Clinical Medicine
VLTU0451c – Theoretical Bases of Clinical Medicine – interactional seminar
5th Semester
1-2 hrs a week, A11/ 114 Campus
3/5 Cancerogenesis
Guarantor of topic: prof. MUDr. Anna Vašků, CSc.
3. 11.2017 A11/114 Campus
8:30-10:10
Dysregulation of blood pressure in hypertension, hypertension in childhood,
methodology of blood pressure measurement –misinterpretation of values (2 hrs- dr. Z.
Nováková)
10.11.2017 A11/114 Campus
8:30-9:20
Dysregulation of blood pressure in hypertension, hypertension in childhood,
methodology of blood pressure measurement –misinterpretation of values (1 hr- dr. Z.
Nováková)
17. 11. 2017 A11/114 Campus Bank Holiday
24. 11.2017 A11/114 Campus
8:30-10:10
Clinical and pharmacological aspects of hypertension II (2 hrs, prof. Souček)
1.12.2017 A11/114 Campus
8:30-10:10
Strokes I (2 hrs, prof. Smrčka)
8.12.2017 A11/114 Campus
8:30-9:20
Strokes II (1 hr, prof. Smrčka)
Theoretical Bases of Clinical Medicine
VLTU0451c – Theoretical Bases of Clinical Medicine – interactional seminar
5th Semester
1-2 hrs a week, A11/ 114 Campus
4/5 Hypertension
Guarantor of topic: prof. MUDr. Miroslav Souček, CSc.
„Read and Write genome“ concept
 Genomes are DNA databases containing coding and
formatting sequences that permit heritable transmission
of the capacity to synthesize biologically adaptive RNA
and protein molecules. In the course of evolution, the
coding sequences and formatting signals change to
encode novel adaptations. Traditionally, these genome
changes have been attributed to accidental causes. But
research on the mechanisms of DNA mutability has led
to a different picture of active biologically mediated
genome restructuring (Shapiro, 2011 and Shapiro,
2013).
 The fluid read–write (RW) genome replaces the constant
read-only memory (ROM) genome.
„Read and Write (RW) genome“
concept
 The basic idea of the RW genome is that cells
use DNA as a modifiable data storage medium
to encode the RNA and protein molecules they
need to deal with changing circumstances.
 Change is continual for living organisms.
Alterations occur as cells progress through the
cell cycle, as environmental conditions vary, as
cells experience damage, as multicellular
morphogenesis proceeds, and as they interact
with other cells and organisms.
„Read and Write (RW) genome“
concept
 To manage recurring short-term variations, cells primarily deploy transient
nucleoprotein complexes to regulate expression of genome data and carry
out the necessary tasks of cell growth and replication.
 For longer-term changes, such as cellular differentiation and multicellular
morphogenesis, heritable epigenetic modifications of the genome come
into play over a number of cell generations.
 For the longest-term changes that create new biological functions in
evolution, cells engage their natural genetic engineering (NGE) capabilities
to acquire and alter DNA sequences and reconfigure genome organization.
Sometimes, NGE functions also alter DNA structure to meet shorter-term
needs, such as rapid generation of diversity among adaptive immune
system receptors and countervailing variation of surface antigens on
microbial invaders.
„Natural genetic engineering
systems“
 The phrase “natural genetic engineering” (NGE)
reflects the reality that living cells possess all the
biochemical tools necessary for cutting, splicing,
polymerizing and modifying DNA (i.e., writing on their
genomes.
 NGE functions are analogous to the tools we use in
artificial genetic engineering. In addition, there are
certain complexes of biochemical activities and DNA
sequences that constitute specialized genome
change cassettes, summarized under the title “mobile
DNA”(Craig et al., 2015).
NGE
Endo- and exo-nucleases
Ligases
Error-free and mutator DNA polymerases
Site-specific recombinases
Homologous recombination complexes
Non-homologous end-joining (NHEJ) complexes
Reverse transcriptases (RTs)
Transposons and transposases
Retroviruses / retrotransposons and cDNA integrases
RNA chaperones / endonucleases and RTs for target-primed reverse transcription (TPRT)
and genome insertion of cDNA copies of retroelement or cellular RNAs
DNA secretion and uptake systems
RNA secretion and uptake systems
Viruses
RW concept and cancer
 Cancer occurs when cells decouple from an
organismʼs normal regulatory apparatus and
proliferate uncontrollably to form a neoplasm,
which may spread around the body to
colonize remote organs (metastasis).
 Cancer remains deeply mysterious. Shapiroʼs
attempt to redefine the field of genomics has
the potential to transform the conceptual
basis for understanding cancer and thereby
suggesting more effective therapies.
RW concept and cancer
 Cancer comes in many varieties (it is, for example,
specific to the organ of origin), but its progression
generally follows a pattern. Clinicians have identified a
dozen or so “hallmarks” of cancer, such as genome
instability and heterogeneity, a shift in metabolism from
oxidation–phosphorylation to fermentation (glycolysis),
disabling of apoptotic pathways, transition to a motile
state, evasion of the immune system, and a degree of
cell–cell cooperation including the recruitment of normal
cells in the vicinity of the primary tumor and in metastatic
niches.
RW concept and cancer
 In spite of the focus on cancer as a human disorder, it is found to be
widespread in biology, occurring among mammals, fish, reptiles and
even plants. This suggests ancient evolutionary roots.
 Multicellular organisms outsource their heritability to specialized
germ cells, and in return somatic cells are subject to strict controls
over their proliferation, such as apoptosis and tumor suppressor
mechanisms.
 Cancer represents a breakdown in these controls, and thus an
abrogation of an ancient covenant that dates back to the dawn of
multicellularity over 1 billion years ago. Even “primitive” organisms
such as sponges, with lineages hundreds of millions of years old,
possess tumor suppressor genes.
Cell division dynamics during lifetime
 Cell division is carefully controled to serve to actual
needs of the organism.
 Early in life, cell division capacity outweighs their
destruction, in adulthood, it is in dynamic equilibrium
and in senescence, involution dominates.
 Some cells are able to obviate the replication
control. They change their phenotype to tumor ones.
 Benign tumor cells
 Malignant tumor cells (invasivity, metastatic
potential).
Cancer
 Cancer does not seem to be a series of
genetic accidents, but a deeply preprogrammed,
ancient, systematic response to
an insult or stress!
 The “cancer subroutine” is unlikely to be as
simple as a cassette of genes activated in an
orderly sequence but a gene regulatory
network with a pre-defined dynamical
trajectory.
Tumorigenesis is a multistep evolutionary process involving myriad,
genetic, and epigenetics alterations. Epigenetic alterations during
cancer initiation and progression provide an abundant source of
variability that not only promotes rapid tumor adaptation but also
provides a rich source of labile biomarkers to complement genetic
analysis
Tumour Biol. 2016 Jul 28. Biomarkers of genome instability
and cancer epigenetics. Reis AH, Vargas FR, Lemos B.
Genome stability
 Since our genomes are constantly
exposed to exogenously-derived (e.g.
UV radiation) and endogenouslyderived
(e.g. metabolically generated
reactive oxygen species) DNA
damaging agents, an impaired ability
to detect and/or respond
appropriately to these efects can
impact on the maintenance of
genetic stability.
O´Driscoll M: Curr Genomics. 2008 May; 9(3): 137–146
Genome stability
 There are many examples of
human Mendelian disorders
defective in the repair of or
response to DNA damage.
 The importance of these pathways
is demonstrated by the increase in
cancer predisposition and
developmental abnormalities
associated with these conditions.
O´Driscoll M: Curr Genomics. 2008 May; 9(3): 137–146
Copy number variants
 The changes in gene copy number are associated with
different phenotypes in humans. Perhaps, the most well
known example of this is the trisomy 21 causative of Down
syndrome.
 An increased expression of the genes on chromosome 21
results directly or indirectly in a clinically heterogeneous
disorder incorporating cognitive impairment, facial
dismorphology, growth retardation, cancer predisposition,
microcephaly, heart and skeletal abnormalities
O´Driscoll M: Curr Genomics. 2008 May; 9(3): 137–146
Gene mutations as a cause
of variability in genes
 Rare alleles (prevalence less than 1% in
population as a result of selection pressure
and/or „recent“ mutation). These mutations
represent „great genetic factors“ causing
monogenic diseases
 Polymorphisms (prevalence more than 1% in
population, smaller genetic factors in
interactions with environmental factors
conditioning complex diseases (including
most of sporadic cancers).
MR Stratton et al. Nature 458, 719-724 (2009)
The lineage of mitotic cell divisions from the
fertilized egg to a single cell within a cancer showing
the timing of the somatic mutations acquired by the
cancer cell and the processes that contribute to
them.
Somatic cell mutations: an example
Sporadic colorectal cancer
MR Stratton et al. Nature 458, 719-724 (2009)
Figurative depiction of the landscape of
somatic mutations present in a single cancer genome.
Somatic cell mutations: an example
Sporadic colorectal cancer
Tumor phenotype
 Is characterised by the process of malignant
transformation of the cell with
- loss of control of cell division (alterations in
cell cycle, antiapoptotic state, „immortality „
of tumor cell, alteration in signal transduction),
- loss of cell-cell contant inhibition, invasivity,
- changes in metabolism
- proangiogenetic activation
 Malignant transformed cells seem to be
resistent to external stress factors as hypoxia,
low pH, hypoglycemia, malnutrition.
Malignant transformed cells
 Continue their division.
 Needs for presence of hormones and
growth factors decreased.
 Production of own growth factors
(autocrinnal stimulation).
 Decrement of ability to stop the growth.
 Decreased ability to stop the growth in
worse nutrition conditions-
Malignant transformed cells
 Occur most frequently in the tissues with quick
proliferation
 Environmental factors have a great impact on gene
expression of the target cells
 A plenty of signals accepted by the cell leads to
activation of specific transcription factors which
decide about either
 division or
 differenciation or
 apoptosis
Tumors and immortality of cells
 Increased activity of telomerase.
 Telomers are key stabilisation factors of terminal part
of the chromosome.
 Telomeres have repetitive sequence TAGGG.
 The length of telomers is decreased after multiple
divisions of the cell (1 cell cycle is shortening of 1
telomere).
 Their renewal is catalysed by telomerase.
Two mechanisms during tumorigenesis:
mechanisms TERT („telomerase reverse transcriptase“)
„alternative lengthening of telomere (ALT) pathway“),
Metastatic phenotype
 Increased motility, invasion and ability
to form metastases.
 Increased production of receptors
(adhesive molecules)
 Increased production of hydrolytic
enzymes
 Function of chemokines
Metastatic phenotype
 Loss of dependence to adhere to
substrates (tumor cells are able to
divide even in tissue culture)
 Loss of gap junctions.
 Changes in cell membrane (glycolipids
and glycoproteins modifications).
Phenomenon of tumor suport
 Chemical cancerogenes:
 Initiators: compounds with cancerogenic
potential after their metabolisation (P450).
 Promoters: effects possible after initiators
(phorbolester).
Tumor genetics
 The development of cancer is associated with a fundamental genetic
change within the cell. Evidence for the genetic origin of cancer is based
on the following:
 Some cancers show a familial predisposition.
 Most known carcinogenes act through induced mutations.
 Susceptibility to some carcinogens depends on the ability of cellular
enzymes to convert them to a mutagenic form.
 Genetically determined traits associated with a deficiency in the
enzymes required for DNA repair are associated with an increased risk of
cancer.
 Some cancers are associated with chromosome 'instability' because of
deficiencies in mismatch repair genes.
 Many malignant tumours represent clonal proliferations of neoplastic
cells.
 Many tumours contain well-described cytogenetic abnormalities, which
involve mutated or abnormally regulated oncogenes and tumour
suppressor genes with transforming activity in cell lines.
 Mutations may occur in the germline and therefore be present in every
cell in the body, or they may occur by somatic mutation in response, for
example, to carcinogens, and therefore be present only in the cells of
the tumour.
Tumor genotype
Mutations in
 Proto-oncogens
 Tumor-supressor genes
 Genes for genome stability
 Genes - modifiers.
Tumor genetics
 There is a small group of autosomal dominant
inherited mutations such as RB (in retinoblastoma)
and a small group of recessive mutations.
 Carriers of the recessive mutations are at risk of
developing cancer if the second allele becomes
mutated, leading to 'loss of heterozygosity' within
the tumour although this is seldom sufficient as
carcinogenesis is a multistep process.
Tumor genetics
 Malignant transformation may result from a gain
in function as cellular proto-oncogenes
become mutated, (e.g. ras), amplified (e.g.
HER2), or translocated (e.g. BCR-ABL).
 Alternatively, there may be a loss of function of
tumour suppressor genes that normally suppress
growth and differentiation.
 However, these mutations are insufficient to
cause malignant transformation by themselves.
Gene mutations as a cause of variability in genes
 Mutations in somatic cells
 are generating in somatic cells during the lifetime
 are cell and/or tissue specific, without transfer to
offspring
 Mutations in germ cells
 they become components of genetic predisposition
 they are present in all cells of the individual
 they are transfered to offspring
Proto-oncogenes and oncogenes
 Up to 100 of various human
protooncogenes in each somatic cell.
 Targets for transduction signals
(mitogenic), by which their expression is
regulated
Proto-oncogenes and oncogenes
 Mutations of proto-oncogenes change
them to oncogenes.
 Dominant mutation.
 Different pathways of proto-oncogenes
activation:
 (a) viral transduction (e.g. onkogene src)
 (b) gene amplification (myc, abl),
 (c) viral insertion (myc)
 (d) chromosomal alteration (myc, abl)
 (e) mutation (ras)
Types of oncogenes
 They are classified to 5 classes:
 (1) growth factors
 (2) growths factors receptors
 (3) intracellular transducers of the signals
 (4) nuclear transcription factors
 (5) proteins controlling cell cycle
Tumor-supresor protein p53 (TP53)
 Is a nuclear protein with key role between
G0 and G1 phase of the cell cycle.
 Mutant variants of the p53 gene can be found
in many cancer types.
 Somatic mutations-usually
 Germ mutations (syndrome Li-Fraumeni).
p53-germ cell mutation
 Li-Fraumeni syndrome (LFS) is a hereditary
cancer predisposition syndrome that is
commonly associated with a germline
mutation in the tumor suppressor gene p53.
 Loss of p53 results in increased expression
of CD44, a cancer stem cell (CSC) marker,
which is involved in the scavenging of
reactive oxygen species (ROS).
Tumor epigenetics
 It is now generally accepted that human cancer
cells harbor global epigenetic abnormalities and
that epigenetic alterations may be the key to
initiating tumorigenesis(Baylin and Jones, 2011;
Sandoval and Esteller, 2012; Sharma et al.,
2010).
 The cancer epigenome is characterized by
substantial changes in various epigenetic
regulatory layers.
 Differences in DNA and histon methylation and
acetylation state of histones in cancer DNA have
been found
Inflammation and tumorigenesis
 Epigenetic switches play an important role in cancer
development.
 Most of the currently discovered switches are induced by
inflammation or involve inflammatory signaling.
 Chronic infections and inflammation contribute to about
25 % of all cancers.
 In many examples, the same inflammatory trigger (e.g.,
IL-6) that induced the switch is constitutively activated in
cancer cells to maintain the new (tumor) phenotype.
Inflamation and tumorigenesis
 This suggests that inflammatory signaling,
which initially originates from immune cells is
adopted by cancer cells through epigenetic
switches. Since epigenetic switches are
reversible, they might represent excellent
targets for anticancer therapy.
Apoptosis and tumorigenesis
 Overexpression of Bcl2 (B-cell lymphoma 2) gene
was found to be related to inhibition of apoptosis
during tumorigenesis.
Tumor neoangiogenesis
 Dysequilibrium between antiangiogenetic and
proangiogenetic factors („angiogenetic switch“).
 Without neoangiogenetic switch, the tumor growth is
possible only to 1 – 2 mm3, when O2 and nutrients
support is possible by difussion from surrounding
tissue.
 Hypoxia of tumor cells
 HIF- alpha induced transcription angiogenetic
factors
Chen L. et aL. Exp Mol Med. 2009; 41(12): 849–857.
Hypoxia and tumor angiogenesis
 Hypoxia occurs in chronic and acute vascular diseases and
tumor formation.
 It is toxic to normal cells, but cancer cells can survive and
continue to proliferate in hypoxia.
 Human tumors grew like cords around blood vessels, and tumor
cells located > 180 µm from the blood vessels were observed to
become necrotic.
 Hypoxia induces angiogenesis during tumor growth; after tumor
growth progresses, an oxygen gradient develops from the
oxygen source to the periphery of the tumor. Cells lacking
oxygen and nutrition because of their distance from the blood
supply become necrotic, whereas those closer to the blood
supply begin sensing hypoxia and secrete angiogenic factors. As
a result, angiogenesis occurs and the tumor develops its own
vasculature, independent of the original tissue.
Thank you for your attention