Molecular Genetics

& Cancer Biology

Meshari Alzahrani
R1 Urology Resident | KAMC-NGHA-Jeddah
16 November 2014
 Lecture Roadmap
 Introduction
 Basic Molecular Genetics
 Tumor Suppressor Genes and Oncogenes
 The Cell Cycle
 DNA Methylation
 DNA Damage and Repair
 Chromosomal Abnormalities and Genetic Instability
 Telomeres and Telomerase
 Apoptosis
 Stem Cells and Cancer
Why This Lecture !
Despite decades of intensive biomedical
research, cancer remains a significant
cause of morbidity and mortality
worldwide.
Campbell-Walsh Urology 10th Edition
 World Cancer Day – 4th February 2014

By 2025, there will be more than 20 million new

cancer cases per year, compared with
14.1 million in 2012, according to the World Cancer
Report 2014, released on 3 February by the World Health
Organization’s International Agency for Research on Cancer.

IARC World Cancer Report 2014

IARC World Cancer Report 2014
cancer ribbon
• However, significant advances in the diagnosis
and treatment of certain Genitourinary (GU)
cancers have been made.
• For example, the cure rate for testicular
cancer now approaches 100%.
(Einhorn, 2002; Horwich et al, 2006)

• Unfortunately, this cancer is unusual in its

responsiveness to therapy and is relatively
uncommon
Campbell-Walsh Urology 10th Edition
• We have had less success with the more
prevalent GU malignancies, such as prostate,
bladder, and renal cancers—the second,
eighth and tenth most common cancers,
respectively.
Campbell-Walsh Urology 10th Edition
 In Saudi Arabia, prostate cancer is the 6th most
common cancer among men of all ages and the most
common cancer among men over the age of 75.
 It accounts for 6.1% of all newly diagnosed cases
among males in year 2010 with an age - standardized
incidence rate of 5.5/100,000 among the male
population.
 Stage at the time of diagnosis is localized in 43.9% of
cases with the remainder being either locally
advanced, metastatic or unknown.
Saudi Cancer Registry Annual Report, 2010
 Bladder cancer ranked 13 among the most common
cancer diagnosis in Saudi Arabia, affecting
3.6/100,000 men and 1/100,000 women.
 In 2010, there were an estimated 243 new cases of
bladder cancer accounting for 2.5% of all newly
diagnosed cases.
 It affected 193 (78.4%) males and 50 (21.6%) females
with a male : female ratio of 385:100.
 The most common histological subtypes is TCC (82%)
followed by SCC(4%).
Saudi Cancer Registry Annual Report, 2010
Basic Molecular Genetics
 In 1953
Co-discoverer of the structure of the DNA molecule

“ The molecular characteristics of DNA

were first described in 1953
(Watson and Crick, 1953).
Francis Crick This molecule serves as the blueprint
“
for determination of structure and
function of all living organisms.

James Watson
 Basic Molecular Genetics
 Somatic cell is any biological cell forming the body of an organism
 Germ cells are cells that give rise to gametes
 Gametes : is a cell that fuses with another cell
during fertilization (conception) in organisms that sexually
reproduce, witch carry half the genetic information of an individual.
 Stem cells are cells that can divide through mitosis and differentiate
into diverse specialized cell types.
 Basic Molecular Genetics
• somatic cells contain DNA arranged
in chromosomes.
• If a somatic cell contains chromosomes arranged
in pairs, it is called diploid and the organism is
called a diploid organism.
• The gametes of diploid organisms contain only
single unpaired chromosomes and are
called haploid.
• Each pair of chromosomes comprises one
chromosome inherited from the father and one
inherited from the mother
 Basic Molecular Genetics
• In humans, somatic cells contain 46 chromosomes
organized into 23 pairs.
• By contrast, gametes of diploid organisms contain
only half as many chromosomes.
• In humans, this is 23 unpaired chromosomes.
• When two gametes (i.e. a spermatozoon and an
ovum) meet during conception, they fuse together,
creating a zygote.
• Due to the fusion of the two gametes, a human
zygote contains 46 chromosomes (i.e. 23 pairs)
 Basic Molecular Genetics
• Chromosome: A distinct segment of linear DNA
containing a large number of genes. In humans
there are 23 such segments, each containing
hundreds to thousands of genes.
• Gene: A segment of DNA that contributes to the
formation of a protein, including both the introns
(noncoding regions) and the exons (coding
regions), as well as the regulatory regions
preceding and following the coding regions.
Mitosis & Meiosis
 Basic Molecular Genetics
 DNA is composed of 3 basic components:
Base : Pyrimidine or purine
Sugar : (2-deoxyribose)
Phosphate
 The Nucleic Acid alphabet consists of 4
bases:
1. purines adenine (A)
2. purines guanine (G)
3. pyrimidines thymine (T)
4. pyrimidines cytosine (C).
5. There is a fifth base that can be
found in DNA known as 5-
nucleoside methylcytosine (5-mC).
 Uracil (U) is substituted for thymine
in the case of RNA.
 The combination of a base and a
sugar (deoxyribose) is referred to as
a nucleoside
DNA : deoxyribonucleic acid

Nucleobases
 Hydrogen bonding occurs specifically between
the purine adenine (A) and the pyrimidine
thymine (T) and between the purine guanine
(G) and the pyrimidine cytosine (C)
 The connection between repeating
phosphates and sugars creates a Helical
Chain.
 In the RNA molecule, adenine base pairs with
uracil (U).
 The combination of a sugar phosphate
group and a base constitutes a
nucleotide.
 The double helix is made from two
polynucleotide chains, each of which
consists of a series of 5′– to 3′–sugar
phosphate links that form a backbone
from which the bases protrude.
 The double helix maintains a constant
width because purines always face
pyrimidines in complementary A-T and G-
C base pairs, respectively.
 Transcription
Transcription is the first step in converting DNA into protein
↓
During the process of transcription, linear DNA is converted to linear messenger RNA (mRNA)
↓
The process of translation consists of the conversion of linear mRNA to a linear set of
amino acids that will eventually form a functional protein
↓
Single strand of RNA is copied from one of the strands of DNA.
↓
The sugar element in the RNA molecule is ribose and the pyrimidine uracil substitutes for thymine
↓
RNA polymerase II is the enzyme that synthesizes the first copy of RNA
This primary strand of RNA is called heterogeneous nuclear RNA (hnRNA)
↓
hnRNA contains coding sequences (exons) of DNA and noncoding sequences (introns).
RNA : ribonucleic acid
During transcription, a section of
one DNA strand, or the other, is
used as a template for the
synthesis of mRNA.
This synthesis always occurs in a
5′ to 3′ direction.
 Protein Translation
 Translation of mRNA into protein occurs in the
cytoplasm where ribosomes are located.
 Two other forms of RNA are important for
protein translation: transfer RNA (tRNA) and
ribosomal RNA (rRNA)
 The mRNA message is translated in segments
of three adjacent nucleotides called a codon
 Each codon is translated into one of 20 amino
acids
 Key Points

The information contained in DNA is

transcribed into RNA and then translated into
protein.
Transcription of RNA is tightly regulated and is
tissue specific.
A single gene can encode for multiple unique
proteins by including or excluding certain exons
in the mRNA transcript by alternative splicing.
Post-transcriptional gene regulation can occur
by a mechanism involving the expression of
noncoding RNAs that have the capability of
binding to and degrading mRNAs
Summary
 Body cells
↓
loss
↓
cell proliferation
↓
maintaining tissue and organ homeostasis
 How can Normal cell become Cancer Cell ?
1. Genetic instability
2. Autonomous growth
3. Insensitivity to internal and external antiproliferative signals
4. Resistance to apoptosis and other forms of induced cell
suicide
5. Unlimited cell division potential
6. The ability to induce new blood vessel formation , a process
termed angiogenesis.
7. Locally invasive behavior, which uniquely distinguishes
malignant from benign neoplasms.
8. Evasion of the immune system.

(Hanahan and Weinberg, 2000; Solimini et al, 2007; Luo et al,2009).

 In addition, cancer cells need to cope with
various cellular stresses that are byproducts of
their abnormal physiology.
 Finally, many cancers develop an additional,
lethal attribute—the ability to leave the site of
the primary tumor to colonize and thrive in
distant organs or tissues as metastases.

(Hanahan and Weinberg, 2000; Solimini et al, 2007; Luo et al,2009).

Our knowledge of the molecular genetics of
cancer is rapidly expanding, providing new
insights that are just beginning to be
successfully exploited for use in novel
diagnostic, prognostic, and therapeutic
applications.

Campbell-Walsh Urology 10th Edition

Tumor Suppressor Genes & Oncogenes
 Tumor Suppressor Genes
 Tumor suppressor genes (antioncogene)
regulate cellular growth and play a critical role
in the normal processes of the cell cycle.
 These genes are also important for DNA repair
and cell signaling.
 The absence of tumor suppressor gene
function may lead to dysregulation of normal
growth control and malignancy.
 Loss of function of both alleles of a tumor
suppressor gene is typically required for
carcinogenesis.
 This functional loss can occur by :
1. homozygous deletion
2. loss of one allele and mutational inactivation of the
second allele,
3. mutational events involving both alleles,
4. loss of one allele and inactivation of the second allele by
DNA methylation
 “two-hit” hypothesis
• The “two-hit” hypothesis was first proposed
in cases of retinoblastoma, which required
mutations in both alleles for disease
manifestation (Knudson, 1971).
• Specific types of mutations in certain gene
however, may not follow this two-hit rule and
can function as dominant negative mutations
that produce altered protein.
• Mutant protein products have been reported
to inhibit function of normal protein from
unaltered alleles (Baker et al, 1990).
• Mutation of a single allele may result in
haploinsufficiency, causing increased
carcinogen susceptibility as in the case of the
TP27 Kip1 gene (Fero et al, 1998).
 Oncogenes
• Oncogenes (proto-oncogene): are associated with
cellular proliferation and are the mutated form of
normal genes.
• Two oncogenes that have been found to be
overexpressed in a variety of cancers include :
c-MYC and c-MET (Wong et al, 1986; Bottaro et al, 1991).
 Key Point
 Mutations in DNA can lead to changes in protein
function or expression that increase the potential
for cancer initiation, progression, or metastasis.
 Tumor suppressor genes regulate and control
cellular growth.
 Oncogenes promote cell growth.
 Key Point
 Loss of tumor suppressor gene function can
occur primarily by :
(1) homozygous deletion
(2) loss of one allele and mutational inactivation of
the second allele
(3) mutational events involving both alleles
(4) loss of one allele and inactivation of the second
allele by DNA methylation.
 Key Point
 Certain tumor suppressor genes do not follow the “two-
hit” hypothesis and may be inactivated by dominant
negative mutations or haploinsufficiency.
 Proto-oncogenes can be converted to oncogenes by :
(1) mutation of the proto-oncogene resulting in an activated form
of the gene
(2) gene amplification
(3) chromosomal rearrangement.
 The Cell Cycle

the cell cycle takes approximately 24 hours to complete

Hartwell et al,1974
checkpoint mechanisms closely monitor
DNA integrity as well as certain critical cell
cycle events.
If problems are detected (e.g., DNA damage),
the cell cycle will pause to allow repair
(Hartwell and Weinert, 1989).

If repair is not possible, normal cells often will

commit cellular suicide through an active
process termed apoptosis.
 Sequential activation of cyclin-dependent kinase
complex (CDKC, cyclin-CDK) is critical to the orderly
progression of cell replication.
 Many oncogenes and tumor suppressors exert
their effects by interfering with cell cycle
checkpoints and apoptotic pathways, allowing
cancer cells to divide continuously and
accumulate.
 Loss of the ability to respond appropriately to
damaged DNA is particularly dangerous, because
it fosters genetic instability, a key attribute of
cancer cells.
 Loss of DNA damage checkpoint controls results
in an increased mutation rate, accelerating the
mutation of cancer-associate genes, thus
contributing to carcinogenesis and disease
progression.
(Bartek et al, 1999).
Some Oncogenes associate with GU
cancers

Oncogenes Associate with Recourse

c-MYC prostate Ca. (Gil et al, 2005).
c-MET RCC (Pisters et al, 1997)
MET proto-oncogene hereditary RCC (Schmidt et al, 1997).
c-MYC bladder Ca. (Schmitz-Drager et al, 1997)
pRB bladder Ca. (Horowitz et al, 1990)
 Key Point
• The cell cycle consists of an ordered,
unidirectional series of events, the main goal of
which is to replicate the cell’s genome and
partition one copy into each of two resulting
daughter cells.
• The cell cycle is divided up into 4 phases; G1, S,
G2, and M phase.
• The transition from G1 into S is critically
dependent on phosphorylation of the pRB tumor
suppressor protein.
 Key Point
 Mutations in pRB are common in some urologic
malignancies (Bladder Ca.)
 Phase-specific phosphorylation of substrate
proteins by cyclin-dependent kinases (CDK)
orchestrate progression through the cell cycle.
 The activities of CDKs are dependent upon their
association with specific cyclin proteins.
 Cyclins accumulate and are rapidly degraded in a
phase-specific manner, thus assuring the proper
sequencing and irreversibility of key events
throughout the cell cycle.
 Key Point
 Primary points of cell cycle control are the G1S
and G2M checkpoints.
 Checkpoints employ cyclin-dependent kinase
inhibitor proteins (CDK1) to pause the cell
cycle in response to a variety of stress signals,
including DNA damage, cell– cell contact,
cytokine release, and hypoxia.
 Key Point
 The TP53 tumor suppressor protein is a key
player in cell cycle checkpoints, responding to
DNA damage by signaling cell cycle arrest and
repair of the damage.
 If the DNA damage cannot be repaired, TP53
may trigger cell death (apoptosis).
 Key Point
• TP53 is the most commonly mutated gene in
cancer and plays a prominent role in
genitourinary malignancies.
• Defects in cell cycle checkpoints lead to
unregulated cell proliferation and genetic
instability.
 DNA Methylation
• The covalent modification of the C-5 position
of cytosine is mediated by DNA (cytosine-5)
methyltransferase, resulting in the formation
of 5-methylcytosine.
• Methylation of cytosine occurs primarily at
the CpG palindrome in DNA.
• One important role for methylation is genomic
imprinting, which results in monoallelic gene
expression without altering the genetic
sequence.
• Loss of imprinting (LOI) is a reduction in the
methylation of the normally methylated allele,
which can lead to activation of the normally
silent copy of a growthpromoting gene.

(Feinberg and Tycko, 2004).

• Changes in global levels and regional patterns
of DNA methylation are among the earliest
and most frequent events known to occur in
human cancer. (Jones and Baylin, 2002)
• Three major pathways by which DNA methylation
may result in genetic dysregulation in human
caner include
(1) inherent mutational effects of 5-methylcytosine
(2) epigenetic effects of promoter methylation on gene
transcription,
(3) potential gene activation and induction of
chromosomal instability by DNA hypomethylation

(Gonzalgo and Jones, 1997).

 DNA Methylation and Prostate Cancer

Abnormal Mutation % Recourse

methylation gene
GSTP1* CpG island >90% PCa* (Lee et al, 1994)
≈ 70% PIN*
RASSF1A* RASSF1A 60% to 70% (Kuzmin et al, 2002)

GSTP1 glutathione-S-transferase Pi
RASSF1A RAS association domain-containing protein 1
Pca Prostate Cancer
PIN prostatic intraepithelial neoplasim
 Role of DNA Methylation
in Bladder Cancer
Abnormal Mutation % Type Recourse
methylation
gene
TP53 C→T 24 urothelial dysplasia (Greenblatt et al, 1994)
transition carcinoma (Rideout et al, 1990;
in situ (CIS) Tornaletti and Pfeifer, 1995).
invasive bladder Ca (Spruck et al,1994b)
TP16 TP16 allele 27-60 primary urothelial (Chan et al, 2002; Chang et
carcinomas al,2003)
CDH1 Hypermeth - high-grade urothelial (Graff et al, 1995; Horikawa et
ylation carcinoma, al, 2003)
RASSF1A RASSF1A 97 primary bladder tumors (Lee et al, 2001)
High tumor grade, (Maruyama et al, 2001).
nonpapillary
growth pattern
muscle invasive disease
DNMT1 Hypomethy Bladder Ca. (Kimura et al,2003)
DNMT3A, lation (Jurgens et al, 1996).
DNMT3B
 Key Point
 Methylation occurs specifically at CpG dinucleotides in
the genome.
 The presence of 5-methylcytosine in DNA can result in
spontaneous deamination to thymine and formation of
C→T transition mutations.
 DNA methylation can affect gene function by
mutational events or epigenetic mechanisms.
 Methylation of CpG islands associated with the
promoter region of genes may result in down-
regulation of transcription and suppression of gene
expression.
 Loss of methylation of normally methylated genes can
lead to the potential for gene expression.
 DNA damage & repair
 DNA damage does not often lead to malignancy,
because the cell possesses multiple repair
mechanisms.
 Defects in DNA repair facilitate the accumulation
of the mutations critical for tumor formation and
progression.
 The cell cycle and the DNA damage response
(DDR) are closely integrated. In response to DNA
damage, the first step is to arrest the cell cycle so
that the DNA can be repaired. TP53 plays a key
role at this interface.
Key Point
 Nucleotide excision repair (NER) is a major
defense against
 DNA damage caused by ultraviolet radiation
and chemical exposure.
 Base excision repair (BER) repairs damage
caused by spontaneous
 deamination of bases, radiation, oxidative
stress alkylating agents, and replication errors.

Key Point
 Mismatch repair (MMR) removes nucleotides
mispaired by DNA polymerase.
 Double-stranded break repair (DSBR) is a
major defense against DNA damage caused by
ionizing radiation, free radicals, and chemicals.
 Many syndromes involving inherited defects in
DNA repair exhibit marked increases in cancer
susceptibility; strongly linking genomic
instability and cancer.

Key Point
Chromosome Abnormalities &
Genetic Instability
 The chromosomal changes seen in solid
tumors can be broken down into two main
classes:
1. changes in the number of whole
chromosomes
2. changes in chromosomal structure.
 Specific Chromosomal Rearrangements
in Genitourinary Malignancies
Recurrent Gene Rearrangements
Cancer oncogene
Pca •ETS transcription
factor family members
RCC MITF/TFE family translocation
carcinomas
Testicular Cancer short
arm of chromosome 12
Hereditary Prostate Cancer HPC families
Sporadic Prostate Cancer chromosome 8
sporadic ccRCC germ line mutations
VHL gene
von Hippel-Lindau syndrom
Bladder Ca. transitional cell chromosome 9
typ (urothelial cell RAS family
carcinomas)
Recourse
Tomlins and
colleagues (2005),
(Hemesath et al, 1994; Argani et
al, 2005).
(Atkin and Baker, 1982; Rodriguez
et al,
1993; Rosenberg et al, 1998;
Verdorfer et al, 2004).
(Smith et al, 1996).
(Gnarra et al, 1994; Shuin et al,
1994).
(Tsai et al, 1990; Cairns et al, 1993;
Linnenbach
et al, 1993)
 Cancer Risk factor
HPC Family history is one of the strongest prostate cancer
risk factors
Bladder Cancer • first degree relatives of patients with bladder cancer are at
increased risk of developing the disease
• high-risk families are very rare and lack clear mendelian
inheritance patterns, precluding classical linkage analysis.
• Bladder cancer is therefore not considered a familial
Disease
 Key Point
 structural rearrangements, as well as
intratumoral heterogeneity in these
aberrations, are hallmarks of most human
solid tumors.
 The extent of chromosomal abnormalities
typically correlates with disease severity and
aggressiveness.
 Recurrent structural rearrangements occur in
prostate (ETS
 Key Point
 family gene fusions), renal (MITF/TFE family
translocation carcinomas), and testicular
cancers (isochromosome 12p).
 Copy number alteration in a particular gene,
coupled with changes in the other allele is
strong evidence for that gene functioning as a
disease-relevant oncogene or tumor
suppressor gene.
 Key Point
 Genes discovered to have germ line
mutations that cause familial forms of cancer
may also be involved in the sporadic form of
the disease (e.g., VHL in ccRCC).
 High-density single nucleotide polymorphism
(SNP) microarrays have been used in genome-
wide association studies (GWAS) to identify
DNA sequence variants associated with cancer
risk.
 Telomere & Telomerase
• Telomeres contain stretches of terminal,
noncoding, repetitive DNA that cap the ends
of each chromosome, thereby stabilizing
them.
• Telomere DNA repeats are progressively lost
as cells divide and as a result of oxidative DNA
damage at the telomeres.
• Normal cells monitor their telomere lengths and
permanently exit the cell cycle (cellular
senescence) or commit suicide (apoptosis) in
response to telomere shortening. This tumor-
suppressive telomere length checkpoint involves
TP53 and pRB.
• Loss of telomere length checkpoints can lead to
critical telomere shortening that initiates
chromosomal instability, thus contributing to
carcinogenesis.
• A majority of cancers and premalignant
lesions have abnormally short telomeres.
• Most cancers express the enzyme telomerase,
which restabilizes the telomeres and allows
unlimited cell division potential
(“immortalization”), thus telomerase
represents an attractive therapeutic target.
 Apoptosis
• Apoptosis:programmed cell death
• Apoptosis is a rapid, orderly, programmed
form of cell death that is used by multicellular
organisms to eliminate unwanted cells.
Through this process, cells are
preprogrammed to commit suicide in
response to various internal and external
signals.
• Apoptosis is believed to play an important role in
tumor suppression because many of the signals that
induce apoptosis arise from potentially tumorigenic
cell stresses such as DNA damage.
• Cancer is characterized by interruptions in the
normal process of apoptosis, resulting in
inappropriate cell survival.
• Apoptosis is mediated by a conserved family
of proteases known as caspases. Initiator
caspases begin caspase proteolytic cascades
that result in the activation of downstream
executioner caspases, which, in turn, target
several cellular proteins.
• Two main apoptotic pathways have been
identified:
In the intrinsic pathway, BCL-2 family members
modulate the release of cytochrome c from
mitochondria, which then participates in the
activation of initiator caspases.
The extrinsic pathway activates caspases in
response to signals from extracellular “death
receptors.”
• In addition to its functions in cell cycle arrest
and DNA repair, TP53 also plays a key role in
apoptosis.
• BCL-2 is a classic inhibitor of the
mitochondrial pathway of apoptosis and is
overexpressed in some genitourinary
malignancies.
• Therapeutic response is often dependent
upon the integrity of apoptotic pathways in
cancer cells. Most TGCT retain intact DDR,
wild-type TP53, and apoptotic responses,
providing high cure rates with DNA-damaging
agents.
• Novel agonists and antagonists of apoptosis,
such as ceramide and clusterin, may be
successfully controlled to combat cancer.
 Cancer Stem Cells
 Stem cells are defined by their ability to
differentiate along multiple lineages and their
immortality.
 Cancer is believed to be a stem cell disease in
which a small population of cancer stem cells
maintains the larger tumor.
 The hedgehog signaling pathway is required
for regeneration of prostate epithelium and
has been implicated in transformation of
prostate progenitor cells.
 Cancer may ultimately be eradicated by
targeting only the cancer stem cell.
 References
• Campbell-Walsh Urology – 10th Edition -
Chapter 18 page 530