Describes various aspects in development of cancer, its molecular biology

1. Molecular Biology of cancer
Dr. Radhakrishna G Pillai
Department of Life Sciences
University of Calicut, Kerala, INDIA

2. Cells and division
• The adult human is composed of approximately
1015 cells
• They divide and differentiate in order to repopulate
organs and tissues which require cell turnover
• Cells composing the epithelial layer of the intestines
which turnover and must be replaced approximately
every 10 days,
• White and red cells life-time varies from 24 h in the
case of some leukocytes to 112 days for mature red
cells
• Cells which have the capacity for division and
replenishment are called stem cells
• There are approximately 1012divisions per day in the
stem cell compartment in bone marrow

3. Controlled division in cells
• Massive proliferation can be initiated by events such as
trauma or infection
• Yet in spite of this enormous production of new cells, the
human body maintains a constant weight over many
decades
• This exquisite control over cell multiplicity is achieved by a
network of overlapping molecular mechanisms which
govern cell proliferation on one hand and cell death, termed
apoptosis
• Apoptosis- cell death by programmed events
• Any factor which alters this balance between birth and
death has the potential to cause cancer if not corrected to
alter the total number of cells in a particular organ or tissue
• After many cell generations this increased cellular
multiplicity would be clinically detectable as neoplasia,
literally new growth

4. Mutations and cancer
• Random mutations in the genes which control prolifer
• Render cells most capable of evading normal
homeostatic mechanismstion or apoptosis are
responsible for cancer
• HeLa cells, derived from a cervical carcinoma which
killed their host in 1956 – successfully used in many
research programs
• The vast majority of mutations that give rise to cancer
are not inherited
• Mutations arise spontaneously as a consequence of
chemical damage to DNA resulting in altered function
of crucial genes

5. HeLa cells
• In a few specific cancers (eg the cervical cancer
that gave rise to HeLa cells) genes encoded by
the HPV virus directly interfere with gene action
and perform the same function as mutations
• Mutations which inactivate these same genes in
non-infected cells have the same carcinogenic
consequences
• Thus parallel evolution also occurs during the
genesis of a cancer cell

6. Cancer- mutations
Most cancers caused by a change in the genome of a
particular cell
This includes:
point mutations which cause amino acid substitutions

7. Mutations in cancer cells
Frame-shift mutations which either truncate the
protein product or scramble its sequence

8. Molecular events in cancer
Chromosomal imbalance or instability resulting in
amplification
Over-expression or inappropriate expression of a particular
gene
Loss of a gene or its fusion with another gene as a result of
chromosomal breakage and rearrangement resulting in a
chimeric protein with altered function
Epigenetic modifications to DNA eg methylation of cytosine
in CpG islands leading to gene silencing

9. Cancer-molbiol
Developing cancer cells select mutations having two basic functions:
mutations which increase the activity of the proteins they code for; this class of
genes are called oncogenes; or
mutations which inactivate gene function in the case of genes classed as tumor
suppressor genes
Regardless of ultimate effect, the types of chemical damage causing these mutations are
believed identical
Their inhibition would be an effective preventive measure
Several genetic diseases which predispose to cancer have as their origin mutations in genes
whose purpose is to protect DNA from mutational events. Thus the understanding of these
events has direct clinical relevance.

10. Mutations
• DNA replication
and subsequent
cell division is
necessary to
convert chemical
damage to an
inheritable change
in DNA that we
call a mutation
• Mutations require proliferation
• Chemical damage to DNA itself is not a mutagenic
event

11. Chance of mutation
• Multiple checks and balances that exist in cells to limit
inappropriate proliferation,
• With few exceptions, malignant human cells must accumulate
multiple mutations in crucial cellular genes that allow their
autonomous replication and invasion
• Mutation at a particular genetic locus is a relatively rare event
• Even after deliberate chemical damage to a cell in a
laboratory situation, the frequency of mutations at a
particular allele is of the order of 10−6, i.e., only one cell in
one million is mutated.
• Mutation rates in human stem cells may be expected to be of
the order of 10−10/cell division, a very low probability, yet
because of the large number of proliferating stem cells it
appears likely that initiation is a common event and all adults
probably contain many mutated cells.

12. Mutations-cancer
• A successful human cancer cell is required to have mutations in at least five
genes
• Probability of a single cell simultaneously acquiring these mutations is
vanishingly small
• The initiated cells needs to clonally expand until the population increases to
many millions
• This process of clonal expansion must then be repeated so that subsequent
mutations can be amassed and cells become progressively better adapted to an
independent life
• This process is observable clinically as disease progression characterized by an
increased growth rate, acquisition of the ability to invade neighboring normal
tissue and to metastasize
• After application of chemotherapeutic agents, tHEY become progressively drug-
resistant.

13. Environment and cancer
• Chemical or physical agents in the environment can be
carcinogenic and the type of damage and mutations they induce
can act as a molecular fingerprint indicating exposure to these
environmental carcinogens
• It is clear however that many human cancers occur in individuals
without obvious exposure to environmental carcinogens and
many human cancers occur in organs for which no environmental
or genetic causes have yet been identified. It must be deduced
then that spontaneous DNA damage does occur which gives rise
to carcinogenic mutations.
• By understanding the causes underlying the genetic damage that
results in cancer we are in a position to reduce its incidence
• Reduction of cancer rates through focused preventive measures is
possible

14. Induction of spontaneous DNA damage
• Spontaneous DNA mutations can occur directly as a consequence of
errors in replication, or
• indirectly as a consequence of chemical damage to DNA leading to
errors in the correct reading of the damaged DNA by DNA polymerase
during the process of replication
• DNA polymerases have high fidelity and with subsequent
proofreading capabilities
• The direct error rate during normal replication of DNA is of the order
1.3×10−10 mutations/base pair/cell division in a human genome of
approximately 2×109 base pairs
• Thus, in a single stem cell, one miscoding error would be introduced
every 10 divisions
• Because approximately 97% of DNA is non-coding and because of the
redundancy of codon recognition, many base changes do not give rise
to amino acid substitutions.

15. Chemical carcinogens
• DNA is also subject to damage from exogenous agents both chemical
and physical- environmental carcinogens
• Perhaps the earliest example of environmental carcinogenesis was
reported in 1775 and involved tumor induction in workers exposed to
coal tar.
• This lead ultimately to the identification of the polycyclic aromatic
hydrocarbon 3,4-benzpyrene and other polycyclic hydrocarbons in coal
tar and the discovery of their action as skin carcinogens in laboratory
animals
• 2-naphthylamine as a bladder carcinogen.
• Stable chemical carcinogens from nature undergo a process of
metabolic activation by enzymes normally involved in the
detoxification of xenobiotic compounds, to yield highly reactive
chemical species – the electrophiles mentioned above (Miller and
Miller, 1975)

16. Physical carcinogens
• Ionizing radiation, both particulate and photon, and ultraviolet
radiation
• They too produce DNA damage which, as with the chemical
carcinogens, can lead to mutations
• Ionizing radiation can cause direct damage to DNA by causing
single and double-strand breaks to the DNA helix, and can also
induce indirect damage as a consequence of radiolysis of water
to yield free radicals
• Most biologically damaging radiation produces ionizations that
are spaced approximately 2 nm apart – the diameter of the DNA
double helix
• Ultraviolet irradiation, though of insufficient energy to produce
ions, is absorbed by DNA bases and is sufficiently energetic to
induce chemical reactions

17. Physical carcinogens
• The most relevant of these occurs between two
adjacent thymidines in the DNA helix and results in
covalent cross linking to form a cyclobutane-linked
thymine dimer
• This disrupts normal base pairing and presents a
formidable obstacle to DNA polymerase, which if not
repaired can give rise to mutations
• Approximately 90% of skin cancers arise in sun-
exposed areas
• The demonstration that ultraviolet causes DNA
damage and that failure to repair this damage results
in carcinogenesis, was the first unequivocal evidence
that damage to DNA was directly implicated in
human cancer.

18. Escaping host immune response
• The first of these is to alter their characteristics
– Immunoselection; When under attack by the immune
system, tumor cells generate variants missing those
features that tag them for destruction by T-cells, other killer
cells, and antibodies
– this process can lead to tumor cells that lack tumor
antigens which present tumor antigens to immune system
cells
– Co stimulatory molecules, which activate T-cells and signal
may also be absent in tumor cells
• The second tactic that cancer cells use to avoid
immune system attack is to suppress the immune
response
• Tumor cells can effect alterations in the host that
minimize an effective immune response against them

19. Escaping host immune response
• The immunosuppression used by tumor cells could
be specific or nonspecific immunosuppression
• The third tactic used to avoid immune system
attack is when tumors hide from the immune
response
– several sites in the human body where immune
reactions are less effective or absent-eg brain
– tumors at these locations can more easily avoid
immune attack than they could if located elsewhere
inside the body
– In addition, a dense tumor stroma possessing
connective tissue can protect tumor cells from
recognition and destruction by the immune system

20. Escaping immune response
• The fourth tactic used by cancer cells to evade immune
attack is to exploit the immune system's ignorance.
– Tumor cells have the ability to grow without eliciting any
immune response
– By immunizing themselves against tumor antigens, an effective
immune surveilance can be generated- immune attack is not
always activated
• The final tactic used by cancer cells to avoid immune attack
it to outpace the immune response
– Tumor cells have the capacity to proliferate so quickly that the
body's normal immune response is not fast enough to keep the
cancer cell growth in check
• Co stimulatory molecules, which activate T-cells and signal
molecules needed to respond to cytokines (i.e. gamma-
interferon), may also be absent in tumor cells

22. Oncogenes
• Gene that has the potential to cause cancer
• In tumor cells, they are often mutated or
expressed at high levels
• Activated oncogenes can cause cells designated for
apoptosis to survive and proliferate
• Most oncogenes require an additional step, such
as mutations in another gene, or environmental
factors, such as viral infection, to cause cancer
• Many cancer drugs target the proteins encoded by
oncogenes

23. Protooncogenes
• A normal gene that can become an oncogene due
to mutations or increased expression
• The resultant protein may be termed as
oncoprotein
• Proto-oncogenes code for proteins that help to
regulate cell growth and differentiation
• Proto-oncogenes are often involved in signal
transduction and execution of mitogenic signals,
usually through their protein products
• Upon activation, a proto-oncogene (or its product)
becomes a tumor-inducing agent, an oncogene
– Examples include RAS, WNT, MYC, ERK, and TRK

24. Mode of action
• An oncogene may cause a cell to secrete growth
factors even though it does not normally do so
• It will thereby induce its own uncontrolled
proliferation (autocrine loop), and proliferation of
neighboring cells
• It may also cause production of growth hormones
in other parts of the body
• Receptor Tyrosine kinases can cause cancer by
turning the receptor permanently on
(constitutively), even without signals from outside
the cell.

25. RAS
• It is evident that the viral oncogene is very similar to
a cellular gene
• RAS is a small GTPase that hydrolyses GTP into GDP
and phosphate
• RAS is activated by growth factor signaling (i.e., EGF,
TGFbeta) and act like a binary switch (on/off) in
growth signaling pathways
• Downstream effectors of RAS include three mitogen-
activated protein kinases
– Raf a MAP Kinase Kinase Kinase (MAPKKK)
– MEK a MAP Kinase Kinase (MAPKK) and
– ERK a MAP Kinase(MAPK), which in turn regulate genes
that mediate cell proliferation.

26. Activation of oncogene
A normal cellular gene or proto-oncogene, can be
converted to an oncogene and then be transformed
into a cancer cell in two ways.
– infection of a normal cell by a retrovirus
– Upon infection, a retrovirus integrates into a chromosal
site adjacent to a normal proto-oncogene
– And carries the proto-oncogene along in the virus' own
genome (including when the virus undergoes replication)
– After the virus replicates, the attached proto-oncogene
can undergo a mutation and thus become an oncogene
– This oncogene and the virus carrying it can now infect
another new healthy cell

27. Activation of oncogene
• A normal cell can be
transformed by a
proto-oncogene that
has already
undergone a
spontaneous or
induced mutation
• The normal cell is
then transformed
into a cancerous one
• The second mechanism occurs when a normal proto-
oncogene undergoes the process of mutation
Cancer cell division

28. Regulation of oncogenes
Molecular Bucket Brigades
• Cancer cells over stimulate the factors they have to promote for abnormal cell
growth, and also develop methods by which they can ignore or avoid signals sent
by adjacent healthy tissue cell to arrest abnormal cell production and growth
• One prime example of this is molecular bucket brigades
– These inhibitory brigades send messages to normal cells to inhibit production and
growth and from there the messages flow to the cell's nucleus
– These molecular brigades may be disrupted, allowing the cell to ignore normally
potent inhibitory signals present at a cell's surface
• In many types of cancer cells, the components vital to formation and
development of these brigades, specified by tumor suppressor genes, are inactive
or absent all together
• In many types of cancer cells,
the components vital to
formation and development of
these brigades, specified by
tumor suppressor genes, are
inactive or absent all together

29. Growth Factors
• The proliferation and differentiation of normal cells
is tightly regulated by exogenous growth factors
• Many transformed cells with oncogenes can bypass
this requirements in a number of ways
– The cell transformed by the inappropriate expression of
an oncogene which encodes either a growth factor or a
constitutively activated growth hormone receptor
– Activated oncogene can modulate one or more
intracellular signal transduction pathways to obviate the
need for extracellular control
– An oncogene product may either activate the production
of a positive growth factor, or inhibit the expression of a
factor whose normal function is to inhibit cell growth

30. TGF-beta Growth Factor & DPC4 Gene
• Transforming growth factor beta or TGF-beta -observed to
have the ability to halt various types of normal, healthy
cell growth
• Some cancer cells have the capacity to inactivate a gene
that encodes a surface receptor for TGF-beta, thereby
making the cancer cells unaffected by TGF-beta
• In addition, some pancreatic cancer cells can inactivate
the DPC4 gene, whose corresponding protein product can
operate downstream of the cell's growth factor receptor
• Furthermore, several kinds of cancer cells possess the
ability to rid themselves of the p15, p53 etc genes. These
genes codes for protein that normally shuts down the
factors that guide the cell through their regular growth
cycle

31. C-myc - regulation
• In many breast cancer cells, there was an elevated
level of c-myc mRNA, primarily due to increased c-
myc mRNA stability
• The c-myc oncogene is essential for cell growth
• Its expression is activated by many peptide growth
factors such as platelet derived growth factor,
fibroblast growth factor, epidermal growth factor,
and growth hormone
• The activation of c-myc expression by most peptide
growth factors is primarily at a post-transcriptional
level by stabilization of the c-myc messenger RNA

32. C-myc regulation
1. Recents research provide evidence that in receptor-
positive, estrogen-responsive human breast cancer
cells, estradiol activate the c-myc oncogene solely by a
transcriptional mechanism
2. In receptor-negative, estrogen independent breast
cancer cells, there was an elevated level of c-myc
mRNA, primarily due to increased c-myc mRNA stability
3. Different mechanisms of regulation of c-myc oncogene
expression exist in estrogenresponsive and estrogen-
nonresponsive human breast cancers