Chemotheraphy of cancer

CHEMOTHERAPHY OF CANCER
Presented by:- Lingaraj .V. Anawal
M.Pharm 2nd SEM
Department of Pharmacology
H.S.K College of Pharmacy B.G.K1

• Neoplasm or tumour is ‘a mass of tissue formed as a result of abnormal,
excessive, unco-ordinated, autonomous and purposeless proliferation of cells even
after cessation of stimulus for growth which caused it’.
• The branch of science dealing with the study of neoplasms or tumours is called
oncology (oncos=tumour, logos=study).
• Neoplasms may be ‘benign’ when they are slow-growing and localised without
causing much difficulty to the host, or ‘malignant’ when they proliferate rapidly
spread throughout the body and may eventually cause death of the host.
• Carcinogenesis or oncogenesis or tumorigenesis means mechanism of induction of
tumours (pathogenesis of cancer)
• Agents which can induce tumours are called carcinogens (Etiology of cancer).
• In 2016, approximately 1.68 million new cancer cases were diagnosed in the
USA, and nearly 600,000 individuals are expected to die from this disease.
• Cancer is the second most common cause of death in the United States,
accounting for 1 in 4 deaths.
• It is a disease characterized by a defect in the normal control mechanisms that
govern cell survival, proliferation, and differentiation.
2

 SPECIAL CATEGORIES OF TUMOURS
1. Mixed tumours= When two types of tumours are combined in the same
tumour it is called a mixed tumour.
a. Adenosquamous carcinoma is the combination of adenocarcinoma and
squamous cell carcinoma in the endometrium.
b. Adenoacanthoma is the mixture of adenocarcinoma and benign
squamous elements in the endometrium. Carcinosarcoma is the rare
combination of malignant tumour of the epithelium (carcinoma) and of
mesenchymal tissue (sarcoma) such as in thyroid.
c. Collision tumour is the two different cancers in the same organ which do
not mix with each other.
d. Mixed tumour of the salivary gland (or pleomorphic adenoma) is the
term used for benign tumour having combination of both epithelial and
mesenchymal tissue elements.
2. Teratomas= Tumours are made up of a mixture of various tissue types
arising from totipotent cells derived from the three germ cell layers—
ectoderm, mesoderm and endoderm. Most common sites for teratomas are
ovaries and testis (gonadal teratomas). But they occur at extra-gonadal
sites as well, mainly in the midline of the body such as in the head and
neck region, mediastinum, retroperitoneum, sacrococcygeal region etc.
Teratomas may be benign or mature or malignant or immature.
3

3. Blastomas (Embryomas)= Blastomas or embryomas are a
group of malignant tumours which arise from embryonal or
partially differentiated cells which would normally form
blastema of the organs and tissue during embryogenesis. The
tumours occur more frequently in infants and children (under 5
years of age) and include some examples of tumours in this age
group: neuroblastoma, nephroblastoma (Wilms’ tumour),
hepatoblastoma, retinoblastoma, medulloblastoma, pulmonary
blastoma.
4. Hamartoma= Hamartoma is benign tumour which is made of
mature but disorganised cells of tissues indigenous to the
particular organ e.g. hamartoma of the lung consists of mature
cartilage, mature smooth muscle and epithelium. Thus, all
mature differentiated tissue elements which comprise the
bronchus are present in it but are jumbled up as a mass.
5. Choristoma= Choristoma is the name given to the ectopic
islands of normal tissue. Thus, choristoma is heterotopia but is
not a true tumour, though it sounds like one.
4

SIGNAL TRANSDUCTION IN CANCER
5

Etiology and Pathogenesis of cancer
 Chemical carcinogens and chemical carcinogenesis
 Physical carcinogens and radiation carcinogenesis
 Biologic carcinogens and viral oncogenesis.
13

 Chemical carcinogens and chemical carcinogenesis
Stages in chemical carcinogenesis
1. Initiation of Carcinogenesis
2. Promotion of Carcinogenesis
3. Progression of cancer
1. Initiation of Carcinogenesis
 Direct-acting carcinogens
 Alkylating agents mainly various anti-cancer drugs (cyclophosphamide, chlorambucil,
busulfan, melphalan, nitrosourea etc…)
 Acylating agents (acetyl imidazole and dimethyl carbamyl chloride)
 Indirect-acting carcinogens or procarcinogens
 Polycyclic aromatic hydrocarbons Main sources are: combustion and chewing of tobacco,
smoke, fossil fuel (e.g. coal), soot, tar, mineral oil, smoked animal foods, industrial and
atmospheric pollutants. Important chemical compounds are: anthracenes (benza-, dibenza-
, dimethyl benza-), benzapyrene and methylcholanthrene.
 Aromatic amines and azo-dyes b-naphthylamine, Benzidine, Azo-dyes.
 Naturally-occurring products aflatoxin B1, actinomycin D, mitomycin C, safrole, betel
nuts.
 Miscellaneous Nitrosamines and nitrosamides , Vinyl chloride monomer, Metals like
nickel, lead, cobalt, chromium etc
1. Metabolic activation
2. Reactive electrophiles
3. Target molecules
4. The initiated cell 14

2. Promotion of Carcinogenesis
 Promoters of carcinogenesis are substances such as phorbol esters,
phenols, artificial sweeteners and drugs like phenobarbital.
3. Progression
 Progression of cancer is the stage when mutated proliferated cell
shows phenotypic features of malignancy. These features pertain to
morphology, biochemical composition and molecular features of
malignancy. Such phenotypic features appear only when the
initiated cell starts to proliferate rapidly progeny of cells that
develops after such repetitive proliferation inherits genetic and
biochemical characteristics of malignancy.
15

Chemical carcinogens and chemical carcinogenesis
17

 PHYSICAL CARCINOGENESIS
Physical agents in carcinogenesis are divided into 2 groups:
1. Radiation (ultraviolet light)
MOA induction of mutation, inhibition of cell division, inactivation of enzymes, cell death.
formation of pyrimidine dimers in DNA
i. Xeroderma pigmentosum is predisposed to skin cancers at younger age (under 20 years
of age).
ii. Ataxia telangiectasia is predisposed to leukaemia.
iii. Bloom’s syndrome is predisposed to all types of cancers.
iv. Fanconi’s anaemia with increased risk to develop cancer.
Ionising radiation. (X-rays, α, β and γ rays, radioactive isotopes, protons and neutrons)
i) It may directly alter the cellular DNA.
ii) It may dislodge ions from water and other molecules of the cell and result in formation
of highly reactive free radicals that may bring about the damage.
2. Non-radiation physical agents.
Mechanical injury to the tissues or prolonged contact with certain physical agents.
i) Stones in the gallbladder and in the urinary tract having higher incidence of cancers of
these organs.
ii) Healed scars following burns or trauma for increased risk of carcinoma of affected skin.
iii) Occupational exposure to asbestos (asbestosis) associated with asbestos-associated
tumours of the lung and malignant mesothelioma of the pleura .
iv) Workers engaged in hardwood cutting or engraving having high incidence of
adenocarcioma of paranasal sinuses.
v) Surgical implants of inert materials such as plastic, glass etc in prostheses.
vi) Foreign bodies embedded in the body for prolonged duration. 18

 BIOLOGIC CARCINOGENESIS
 Parasites Schistosoma haematobium infection of the urinary bladder
is associated with high incidence of squamous cell carcinoma of the
urinary bladder in some parts of the world such as in Egypt.
 Clonorchis sinensis, the liver fluke, lives in the hepatic duct and is
implicated in causation of cholangiocarcinoma.
 Fungus Aspergillus flavus grows in stored grains and liberates
aflatoxin its human consumption, especially by those with HBV
infection is associated with development of hepatocellular
carcinoma.
 Bacteria Helicobacter pylori, a gram-positive spiralshaped micro-
organism, colonises the gastric mucosa and has been found in cases
of chronic gastritis and peptic ulcer; its prolonged infection may
lead to gastric lymphoma and gastric carcinoma.
19

 VIRAL CARCINOGENESIS
 Viral Oncogenesis
1. Mode of DNA viral oncogenesis.
Host cells infected by DNA oncogenic viruses may have one of the
following 2 results.
i) Replication The virus may replicate in the host cell with
consequent lysis of the infected cell and release of virions.
ii) Integration The viral DNA may integrate into the host cell DNA.
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Mode of DNA and RNA viral oncogenesis
21

2. Mode of RNA viral oncogenesis
 RNA viruses or retroviruses contain two identical strands of RNA and the enzyme
reverse transcriptase.
i) Reverse transcriptase is RNA dependent DNA synthetase that acts as a template
to synthesise a single strand of matching viral DNA.
ii) The single strand of viral DNA is then copied by DNA dependent DNA
synthetase to form another strand of complementary DNA resulting in double-
stranded viral DNA or provirus.
iii) The provirus or double-stranded viral DNA is then integrated into the DNA of
the host cell genome and may induce mutation and thus transform the cell into
neoplastic cell.
iv) Retroviruses are replication competent. The host cells which allow replication of
integrated retrovirus are called permissive cells. Non-permissible cells do not
permit replication of the integrated retrovirus.
v) Viral replication begins after integration of the provirus into host cell genome.
Integration results in transcription of proviral genes or progenes into messenger
RNA which then forms components of the virus particle—virion core protein
from gag gene, reverse transcriptase from pol gene, and envelope glycoprotein
from env gene.
vi) The three components of virus particle are then assembled at the plasma
membrane of the host cell and the virus particles released by budding off from
the plasma membrane, thus completing the process of replication. we now turn
to specific DNA and RNA oncogenic viruses and their specific oncogenic role.
22

MOLECULAR PATHOGENESIS OF CANCER
25

SIX HALL MARKS OF CANCER
26

• The molecular pathogenesis of cancer and the carcinogenic
agents that inflict genetic damage. In the past 20 years
hundreds of cancer-associated genes have been discovered.
such as p53, are most commonly mutated others, c-ABL, are
affected only in certain leukemias.
• Cancer gene has a specific function the dysregulation of
which contributes to the origin or progression of malignancy.
 Self-sufficiency in growth signals
 Insensitivity to growth-inhibitory signals
 Evasion of apoptosis
 Limitless replicative potential (i.e., overcoming cellular
senescence and avoiding mitotic catastrophe)
 Development of sustained angiogenesis
 Ability to invade and metastasize
 Genomic instability resulting from defects in DNA repair
27

1. Self-Sufficiency in Growth Signals
• Genes that promote autonomous cell growth in cancer cells are
called oncogenes. They are derived by mutations in proto-
oncogenes and are characterized by the ability to promote cell
growth in the absence of normal growth-promoting signals. Their
products called oncoproteins resemble the normal products of
proto-oncogenes except that oncoproteins are devoid of important
regulatory elements, and their production in the transformed cells
does not depend on growth factors or other external signals.
• Under physiologic conditions, cell proliferation can be readily
resolved into the following steps:
 Growth Factors
 Growth Factor Receptors
 Signal-Transducing Proteins
 Nuclear Transcription Factors
 Cyclins and Cyclin-Dependent Kinases (CDKs) 28

• The binding of a growth factor to its specific receptor on the cell membrane
transient and limited activation of the growth factor receptor, which in turn
activates several signal-transducing proteins on the inner leaflet of the plasma
membrane. Transmission of the transduced signal across the cytosol to the nucleus
via second messengers or a cascade of signal transduction molecules Induction
and activation of nuclear regulatory factors that initiate DNA transcription. Entry
and progression of the cell into the cell cycle resulting ultimately in cell division.
 Growth Factors
• All normal cells require stimulation by growth factors to undergo proliferation.
growth factors are made by one cell type and act on a neighboring cell to stimulate
proliferation (paracrine action).
• Cancer cells acquire growth self-sufficiency, by acquiring the ability to synthesize
the same growth factors to which they are responsive. For example, many
glioblastomas secrete platelet-derived growth factor (PDGF) and express the
PDGF receptor, and many sarcomas make both transforming growth factor-α
(TGF-α) and its receptor.
• Similar autocrine loops are fairly common in many types of cancer. Genes that
encode homologues of fibroblast growth factors (e.g., hst-1 and FGF3) have been
detected in several gastrointestinal and breast tumors.
• FGF-2 is expressed in human melanomas but not normal melanocytes.
• Hepatocyte growth factor (HGF) and its receptor c-Met are both overexpressed in
follicular carcinomas of the thyroid.
• In many instances the growth factor gene itself is not altered or mutated, but the
products of other oncogenes (e.g., RAS) stimulate overexpression of growth factor
genes and the subsequent development of an autocrine loop. 29

 Growth Factor Receptors
• Several oncogenes that result from the overexpression or mutation of
growth factor receptors have been identified. Mutant receptor proteins
deliver continuous mitogenic signals to cells even in the absence of the
growth factor in the environment.
• The best-documented examples of overexpression involve the epidermal
growth factor (EGF) receptor family. ERBB1, the EGF receptor, is
overexpressed in 80% of squamous cell carcinomas of the lung, 50% or
more of glioblastomas, 80 to 100% of epithelial tumors of the head and
neck.
• A related receptor called HER2/NEU (ERBB2) is amplified in 25% to
30% of breast cancers and adenocarcinomas of the lung, ovary, and
salivary glands.
• These tumors are exquisitely sensitive to the mitogenic effects of small
amounts of growth factors, and a high level of HER2/NEU protein in
breast cancer cells is a harbinger of poor prognosis. The significance of
HER2/NEU in the pathogenesis of breast cancers derived clinical benefit
from blocking the extracellular domain of this receptor with anti-
HER2/NEU antibodies. Treatment of breast cancer with anti-HER2/NEU
antibody is an elegant example of "bench to bedside" medicine.
30

 Signal-Transducing Proteins
• A mechanism by which the signaling molecules couple growth factor receptors to
their nuclear targets. such signaling proteins are associated with the inner leaflet
of the plasma membrane where they receive signals from activated growth factor
receptors and transmit them to the nucleus either through second messengers or
through a cascade of phosphorylation and activation of signal transduction
molecules. Two important members in this category are RAS and ABL.
• RAS is a member of a family of small G proteins that bind guanosine nucleotides
(guanosine triphosphate [GTP] and guanosine diphosphate [GDP]), similar to the
larger trimolecular G proteins. Normal RAS proteins flip back and forth between
an excited signal transmitting state and a quiescent state. RAS proteins are
inactive when bound to GDP stimulation of cells by growth factors leads to
exchange of GDP for GTP and subsequent conformational changes that generates
active RAS. The activated RAS in turn stimulates down stream regulators of
proliferation such as the RAF mitogen activated protein (MAPk) kinase mitogenic
cascade which floods the nucleus with signals for cell proliferation.
31

• The excited signal-emitting stage of the normal RAS protein is
short lived, because its intrinsic guanosine triphosphatase
(GTPase) activity hydrolyzes GTP to GDP releasing a
phosphate group and returning the protein to its quiescent
inactive state. The GTPase activity of activated RAS protein is
magnified dramatically by a family of GTPase activating
proteins (GAPs) which act as molecular brakes that prevent
uncontrolled RAS activation by favoring hydrolysis of GTP to
GDP.
• The RAS gene is most commonly activated by point mutations.
Molecular analyses of RAS mutations have revealed three hot
spots, which encode residues either within the GTP-binding
pocket or the enzymatic region essential for GTP hydrolysis.
Mutations at these locations interfere with GTP hydrolysis that
is essential to convert RAS into an inactive form. RAS is thus
trapped in its activated GTP-bound form, and the cell is forced
into a continuously proliferating state. It follows from this
scenario that the consequences of mutations in RAS protein
would be mimicked by mutations in the GAPs that fail to
restrain normal RAS proteins.
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1. Self-Sufficiency in Growth Signals
33

 Nuclear Transcription Factors
• All signal transduction pathways enter the nucleus and have an impact on a large
bank of responder genes that orchestrate the cells orderly advance through the
mitotic cycle. ultimate consequence of signaling through oncogenes like RAS or
ABL is inappropriate and continuous stimulation of nuclear transcription factors
that drive growth promoting genes.
• Growth autonomy may thus occur as a consequence of mutations affecting genes
that regulate transcription of DNA. Oncoproteins of host including products of
the MYC, MYB, JUN, FOS, and REL oncogenes, function as transcription factors
that regulate the expression of growth-promoting genes such as cyclins. The
MYC gene is involved most commonly in human tumors. The MYC proto-
oncogene is expressed in virtually all cells and the MYC protein is induced
rapidly when quiescent cells receive a signal to divide. In normal cells MYC
levels decline to near basal level when the cell cycle begins.
• The MYC protein can either activate or repress the transcription of other genes.
Those activated by MYC include several growth promoting genes including
cyclin-dependent kinases (CDKs), whose products drive cells into the cell cycle.
Genes repressed by MYC include the CDK inhibitors (CDKIs). MYC promotes
tumorigenesis by increasing expression of genes that promote progression
through the cell cycle and repressing genes that slow or prevent progression
through the cell cycle. Dysregulation of the MYC gene resulting from a
translocation occurs in Burkitt lymphoma a B-cell tumor. MYC is also amplified
in breast, colon, lung, and many other cancers.
34

Cyclins and Cyclin-Dependent Kinases (CDKs)
35

 Cyclins and Cyclin-Dependent Kinases (CDKs)
• All growth-promoting stimuli is the entry of quiescent cells into the cell
cycle. Cancers may become autonomous if the genes that drive the cell
cycle become dysregulated by mutations or amplification the orderly
progression of cells through the various phases of the cell cycle is
orchestrated by CDKs which are activated by binding to cyclins.
• The CDK-cyclin complexes phosphorylate crucial target proteins that
drive the cell through the cell cycle. On completion of this task cyclin
levels decline rapidly. More than 15 cyclins have been identified cyclins
D, E, A, and B appear sequentially during the cell cycle and bind to one
or more CDK.
• The cell cycle may thus be seen as a relay race in which each lap is
regulated by a distinct set of cyclins and as one set of cyclins leaves the
track the next set takes over. The expression of cyclin D or CDK4 seem
to be a common event in neoplastic transformation. The cyclin D genes
are overexpressed in many cancers including those affecting the breast,
esophagus, liver, and a subset of lymphomas.
• Amplification of the CDK4 gene occurs in melanomas, sarcomas, and
glioblastomas. Mutations affecting cyclin B and cyclin E and other
CDKs also occur but they are much less frequent than those affecting
cyclin D/CDK4.
36

• While cyclins arouse the CDKs, their inhibitors (CDKIs), of which there are many,
silence the CDKs and exert negative control over the cell cycle.
• One family of CDKIs, composed of three proteins, called p21 [CDKN1A], p27
[CDKN1B], p57 [CDKN1C], inhibits the CDKs,
• The other family of CDKIs has selective effects on cyclin D/CDK4 and cyclin D/CDK6.
The four members of this family (p15 [CDKN2B], p16 [CDKN2A], p18 [CDKN2C], and
p19 [CDKN2D]) are sometimes called INK4 (A-D) proteins.
• Expression of these inhibitors is down-regulated by mitogenic signaling pathways thus
promoting the progression of the cell cycle. For example, p27 [CDKN1B], a CDKI that
inhibits cyclin E, is expressed throughout G1.
• Mitogenic signals obtund p27 in a variety of ways, relieving inhibition of cyclin E-CDK2
and thus allowing the cell cycle to proceed. The CDKN2A gene locus, also called
INK4a/ARF, encodes two protein products that is p16 INK4A and p14ARF. Both block
cell cycle progression but have different targets.
• p16 [CDKN2A] inhibits RB phosphorylation by blocking cyclin D-CDK4 complex,
whereas p14ARF activates the p53 pathway by inhibiting MDM2.
• Thus both proteins function as tumor suppressors and deletion of this locus frequent in
many tumors impacts both the RB and p53 pathways. The CDKIs are frequently mutated
or otherwise silenced in many human malignancies.
• Germ-line mutations of CDKN2A are associated with 25% of melanoma prone kindreds.
Somatically acquired deletion or inactivation of CDKN2A is seen in 75% of pancreatic
carcinomas, 40% to 70% of glioblastomas, 50% of esophageal cancers, 20% of non-
small-cell lung carcinomas, soft tissue sarcomas, and bladder cancers.
37

2. Insensitivity to Growth-Inhibitory Signals
• Isaac Newton predicted that every action has an equal and opposite
reaction. his formulation holds true for cell growth. Oncogenes
encode proteins that promote cell growth the products of tumor
suppressor genes apply brakes to cell proliferation. Disruption of
such genes renders cells refractory to growth inhibition and mimics
the growth promoting effects of oncogenes.
• The retinoblastoma (RB) gene the first and prototypic cancer
suppressor gene to be discovered. Retinoblastoma an uncommon
childhood tumor 60% of retinoblastomas are sporadic and the
remaining 40% are familial the predisposition to develop the tumor
being transmitted as an autosomal dominant trait. The sporadic and
familial occurrence of an identical tumor, Knudson, in 1974,
proposed his now famous two-hit hypothesis which in molecular
terms can be stated as follows: Two mutations (hits) are required to
produce retinoblastoma. These involve the RB gene, located on
chromosome 13q14. 38

• In familial cases children inherit one defective copy of the RB gene
in the germ line the other copy is normal. Retinoblastoma develops
when the normal RB gene is lost in retinoblasts as a result of
somatic mutation. Retinoblastoma families only a single somatic
mutation is required for expression of the disease the familial
transmission follows an autosomal dominant inheritance pattern. In
sporadic cases both normal RB alleles are lost by somatic mutation
in one of the retinoblasts. The end result is the same a retinal cell
that has lost both of the normal copies of the RB gene becomes
cancerous.
• The loss of normal RB genes was discovered initially in
retinoblastomas it is now evident that homozygous loss of this
gene is a fairly common event in several tumors including breast
cancer, small-cell cancer of the lung, and bladder cancer. Patients
with familial retinoblastoma also are at greatly increased risk of
developing osteosarcomas and some soft tissue sarcomas.
• Antigrowth signals can prevent cell proliferation by two
complementary mechanisms. The signal may cause dividing cells
to go into G0 (quiescence), where they remain until external cues
prod their reentry into the proliferative pool.
39

2. Insensitivity to Growth-Inhibitory Signals (RB Gene and Cell Cycle)
40

 Insensitivity to Growth-Inhibitory Signals (RB Gene and Cell Cycle)
41

• The RB gene product is a DNA binding protein expressed in every cell type. It
exists in an active hypophosphorylated and an inactive hyperphosphorylated state.
• The importance of RB lies in its enforcement of G1 or the gap between mitosis
(M) and DNA replication (S). As development proceeds two gaps are incorporated
into the cell cycle: Gap 1 (G1) between mitosis (M) and DNA replication (S) and
Gap 2 (G2) between DNA replication (S) and mitosis (M) each phase of the cell
cycle circuitry is monitored carefully the transition from G1 to S. Once cells cross
the G1 checkpoint they can pause the cell cycle for a time but they are obligated to
complete mitosis. In G1 cells can exit the cell cycle either temporarily called
quiescence or permanently called senescence. In G1 signals are integrated to
determine whether the cell should enter the cell cycle or exit the cell cycle and
differentiate or die RB is a key node in this decision process.
• Initiation of DNA replication requires the activity of cyclin E/CDK2 complexes
and expression of cyclin E is dependent on the E2F family of transcription factors.
In G1 RB is in its hypophosphorylated active form and it binds to and inhibits the
E2F family of transcription factors preventing transcription of cyclin E.
• Hypophosphorylated RB blocks E2F mediated transcription in at least two ways
1) sequesters E2F preventing it from interacting with other transcriptional
activators. 2)RB recruits chromatin remodeling proteins such as histone
deacetylases and histone methyltransferases which bind to the promoters of E2F
responsive genes such as cyclin E.
42

• These enzymes modify chromatin at the promoters to make DNA insensitive to
transcription factors. This situation is changed upon mitogenic signaling. Growth
factor signaling leads to cyclin D expression and activation of cyclin D-CDK4/6
complexes.
• These complexes phosphorylate RB inactivating the protein and releasing E2F to
induce target genes such as cyclin E. Expression of cyclin E then stimulates
DNA replication and progression through the cell cycle. When the cells enter S
phase they are committed to divide without additional growth factor stimulation.
During the ensuing M phase, the phosphate groups are removed from RB by
cellular phosphatases regenerating the hypophosphorylated form of RB.
• E2F is not the sole target of RB. The versatile RB protein has been shown to
bind to a variety of other transcription factors that regulate cell differentiation.
For example, RB stimulates myocyte-, adipocyte-, melanocyte-, and
macrophage-specific transcription factors.
• RB is central to the control of the cell cycle one may ask why RB is not mutated
in every cancer. Mutations in other genes that control RB phosphorylation can
mimic the effect of RB loss. For example, mutational activation of CDK4 or
overexpression of cyclin D would favor cell proliferation by facilitating RB
phosphorylation and inactivation.
43

p53 Gene: Guardian of the Genome
45

 p53 Gene: Guardian of the Genome
• The p53 tumor suppressor gene is one of the most commonly mutated genes
in human cancers. p53 thwarts neoplastic transformation by three interlocking
mechanisms: 1) activation of temporary cell cycle arrest (termed quiescence)
2) induction of permanent cell cycle arrest (termed senescence) or 3)
triggering of programmed cell death (termed apoptosis). p53 can be as a
central monitor of stress, directing the stressed cells toward an appropriate
response. A stresses can trigger the p53 response pathways including anoxia,
inappropriate oncogene expression (e.g., MYC or RAS) and damage to the
integrity of DNA. the DNA-damage response p53 plays a central role in
maintaining the integrity of the genome.
• healthy cells p53 has a short half-life (20 minutes) because of its association
with MDM2 a protein that targets it for destruction. When the cell is stressed,
for example by an assault on its DNA, p53 undergoes post-transcriptional
modifications that release it from MDM2 and increase its half-life. During the
process of being unshackled from MDM2, p53 also becomes activated as a
transcription factor. The genes whose transcription is triggered by p53 have
been found. can be grouped into two broad categories: those that cause cell
cycle arrest and those that cause apoptosis. If DNA damage can be repaired
during cell cycle arrest the cell reverts to a normal state if the repair fails, p53
induces apoptosis or senescence.
46

• The key initiators of the DNA-damage pathway are two related protein kinases: ataxia-
telangiectasia mutated (ATM) and ataxia-telangiectasia mutated related (ATR). As the
name implies the ATM gene was originally identified as the germ-line mutation in patients
with ataxia-telangiectasia. Patients with this disease, which is characterized by an inability
to repair certain kinds of DNA damage suffer from an increased incidence of cancer. The
types of damage sensed by ATM and ATR are different but the down-stream pathways they
activate are similar. Once triggered both ATM and ATR phosphorylate a variety of targets
including p53 and DNA repair proteins. Phosphorylation of these two targets leads to a
pause in the cell cycle and stimulation of DNA repair pathways respectively.
• p53-mediated cell cycle arrest occurs in the G1 phase and is caused mainly by p53-
dependent transcription of the CDKI CDKN1A (p21). The CDKN1A gene as described
earlier inhibits cyclin-CDK complexes and prevents phosphorylation of RB essential for
cells to enter G1 phase. Such a pause in cell cycling is gives the cells "breathing time" to
repair DNA damage.
• p53 also helps the process by inducing certain proteins such as GADD45 (growth arrest
and DNA damage), that help in DNA repair. p53 can stimulate DNA repair pathways by
transcription-independent mechanisms as well. If DNA damage is repaired successfully,
p53 up-regulates transcription of MDM2, leading to destruction of p53 and relief of the cell
cycle block. If the damage cannot be repaired, the cell may enter p53-induced senescence
or undergo p53-directed apoptosis.
47

• p53-induced senescence is a permanent cell cycle arrest
characterized by specific changes in morphology and gene
expression that differentiate it from quiescence or reversible cell
cycle arrest. Senescence requires activation of p53 and/or RB
and expression of their mediators such as the CDKIs. Such cell
cycle arrest is generally irreversible although it may require the
continued expression of p53. The mechanisms of senescence
involve global chromatin changes, which drastically and
permanently alter gene expression.
• p53 senses DNA damage and assists in DNA repair by causing
G1 arrest and inducing DNA repair genes. A cell with damaged
DNA that cannot be repaired is directed by p53 to either enter
senescence or undergo apoptosis. p53 has been rightfully
called a "guardian of the genome."
• the importance of p53 in controlling carcinogenesis more than
70% of human cancers have a defect in this gene, and the
remaining malignant neoplasms have defects in genes up-stream
or down-stream of p53. loss of the p53 gene is found in every
type of cancer, including carcinomas of the lung, colon, and
breast-the three leading causes of cancer deaths.
48

Transforming Growth Factor-β Pathway
• Although much is known about the circuitry that applies brakes to the
cell cycle, the molecules that transmit antiproliferative signals to cells
are less well characterized. Best known is TGF-β, a member of a family
of dimeric growth factors that includes bone morphogenetic proteins and
activins. In most normal epithelial, endothelial, and hematopoietic cells,
TGF-β is a potent inhibitor of proliferation. It regulates cellular
processes by binding to a complex composed of TGF-β receptors I and
II. Dimerization of the receptor upon ligand binding leads to a cascade
of events that result in the transcriptional activation of CDKIs with
growth-suppressing activity, as well as repression of growth-promoting
genes such as c-MYC, CDK2, CDK4, and cyclins A and E.
• In many forms of cancer, the growth-inhibiting effects of TGF-β
pathways are impaired by mutations in the TGF-β signaling pathway.
These mutations may affect the type II TGF-βreceptor or SMAD
molecules that serve to transduce antiproliferative signals from the
receptor to the nucleus. Mutations affecting the type II receptor are seen
in cancers of the colon, stomach, and endometrium. Mutational
inactivation of SMAD4, one of 10 proteins involved in TGF-β signaling,
is common in pancreatic cancers. In 100% of pancreatic cancers and
83% of colon cancers, at least one component of the TGF-β pathway is
mutated.
49

 Adenomatous Polyposis Coli-β-Catenin Pathway
50

• Adenomatous Polyposis Coli-β-Catenin Pathway
• the rare hereditary disease called adenomatous polyposis coli (APC) patients
develop numerous adenomatous polyps in the colon that have a very high
incidence of transformation into colonic cancers. These patients consistently
show loss of a tumor suppressor gene called APC (named for the disease). The
APC gene exerts antiproliferative effects. It is a cytoplasmic proteinfunction is to
regulate the intracellular levels of β-catenin a protein with many functions. On
the one hand β-catenin binds to the cytoplasmic portion of E-cadherin a cell
surface protein that mediates intercellular interactions on the other hand it can
translocate to the nucleus and activate cell proliferation.
• β-catenin is an important component of the WNT signaling pathway that
regulates cell proliferation. WNT is a soluble factor that can induce cellular
proliferation. It does by binding to its receptor and transmitting signals that
prevent the degradation of β-catenin allowing it to translocate to the nucleus
where it acts as a transcriptional activator in conjunction with another molecule
called TcF. In quiescent cells are not exposed to WNT result in cytoplasmic β-
catenin is degraded by a destruction complex, of which APC is an integral part.
loss of APC (in malignant cells) β-catenin degradation is prevented, and the WNT
signaling response is inappropriately activated in the absence of WNT. This leads
to transcription of growth-promoting genes, such as cyclin D1 and MYC.
• APC behaves as a tumor suppressor gene. Individuals born with one mutant allele
develop hundreds to thousands of adenomatous polyps in the colon during their
teens or 20s. one or more polyps undergo malignant transformation upon
accumulation of other mutations in the cells within the polyp. APC mutations are
seen in 70% to 80% of sporadic colon cancers.
51

3. Evasion of Apoptosis
• Accumulation of neoplastic cells may result not only from activation of growth-
promoting oncogenes or inactivation of growth-suppressing tumor suppressor
genes but also from mutations in the genes that regulate apoptosis.
• A large family of genes that regulate apoptosis has been identified how tumor
cells evade apoptosis. There are two distinct programs that activate apoptosis the
extrinsic and intrinsic pathways the sequence of events that lead to apoptosis by
signaling through the death receptor CD95/Fas (extrinsic pathway) and by DNA
damage (intrinsic pathway).
• The extrinsic pathway is initiated when CD95 is bound to its ligand, CD95L,
leading to trimerization of the receptor and thus its cytoplasmic death domains
which attract the intracellular adaptor protein FADD. This protein recruits
procaspase 8 to form the death-inducing signaling complex. Procaspase 8 is
activated by cleavage into smaller subunits, generating caspase 8. Caspase 8 then
activates down-stream caspases such as caspase 3, a typical executioner caspase
that cleaves DNA and other substrates to cause cell death.
54

• The intrinsic pathway of apoptosis is triggered by a variety of
stimuli including withdrawal of survival factors, stress, and injury.
• Activation of this pathway leads to permeabilization of
mitochondrial outer membrane with resultant release of molecules,
such as cytochrome c, that initiate apoptosis.
• The integrity of the mitochondrial outer membrane is regulated by
pro-apoptotic and anti-apoptotic members of the BCL2 family of
proteins.
• The pro-apoptotic proteins, BAX and BAK are required for
apoptosis and directly promote mitochondrial permeabilization.
• Their action is inhibited by the anti-apoptotic members of this
family exemplified by BCL2 and BCL-XL. A third set of proteins
(so-called BH3-only proteins) including BAD, BID, and PUMA,
regulate the balance between the pro- and anti-apoptotic members of
the BCL2 family.
55

• The BH3-only proteins promote apoptosis by neutralizing
the actions of anti-apoptotic proteins like BCL2 and BCL-
XL. When the sum total of all BH3 proteins expressed
"overwhelms" the anti-apoptotic BCL2/BCLXl protein
barrier, BAX and BAK are activated and form pores in the
mitochondrial membrane. Cytochrome c leaks into the
cytosol, where it binds to APAF-1, activating caspase 9.
Like caspase 8 of the extrinsic pathway, caspase 9 can
cleave and activate the executioner caspases.
• The multiple sites at which apoptosis is frustrated by cancer
cells Starting from the surface, reduced levels of CD95 may
render the tumor cells less susceptible to apoptosis by Fas
ligand (FasL).
• Some tumors have high levels of FLIP a protein that can
bind death-inducing signaling complex and prevent
activation of caspase 8.
56

4. Limitless Replicative Potential
57

4. Limitless Replicative Potential
• Normal human cells have a capacity of 60 to 70 doublings after this the cells lose
the capacity to divide and enter senescence. This phenomenon leads to
progressive shortening of telomeres at the ends of chromosomes. Short telomeres
seem to be recognized by the DNA repair machinery as double-stranded DNA
breaks and this leads to cell cycle arrest mediated by p53 and RB.
• Cells in which the checkpoints are disabled by mutations in p53 or RB. The
nonhomologous end joining pathway is activated as a last ditch effort to save the
cell joining the shortened ends of two chromosomes. Inappropriately activated
repair system results in dicentric chromosomes that are pulled apart at anaphase
resulting in new double-stranded DNA breaks.
• The resulting genomic instability from the repeated bridge fusion breakage cycles
eventually produces mitotic catastrophe characterized by massive cell death.
Tumors to grow indefinitely as they often do loss of growth restraints is not
enough. Tumor cells must also develop ways to avoid both cellular senescence
and mitotic catastrophe. During crisis a cell manages to reactivate telomerase the
bridge-fusion-breakage cycles cease and the cell is able to avoid death. During
crisis of genomic instability that precedes telomerase activation numerous
mutations could accumulate helping the cell march toward malignancy.
• Telomerase active in normal stem cells is normally absent from or at very low
levels in most somatic cells. Telomere maintenance is seen in virtually all types
of cancers.
• In 85% to 95% of cancers this is due to up-regulation of the enzyme telomerase
58

5. Development of Sustained Angiogenesis
• The genetic abnormalities of tumors cannot enlarge beyond 1 to 2
mm in diameter unless they are vascularized. Tumors require
delivery of oxygen and nutrients and removal of waste products,
the 1- to 2-mm zone represents the maximal distance across which
oxygen, nutrients, and waste can diffuse from blood vessels.
• Cancer cells can stimulate neo-angiogenesis during which new
vessels sprout from previously existing capillaries or in some cases
vasculogenesis in which endothelial cells are recruited from the
bone marrow. Tumor vasculature is abnormal. The vessels are
leaky, dilated, and have a haphazard pattern of connection.
• Perfusion supplies needed nutrients, oxygen, and newly formed
endothelial cells stimulate the growth of adjacent tumor cells by
secreting growth factors such as insulin-like growth factors, PDGF,
and granulocyte-macrophage colony-stimulating factor.
• Angiogenesis is required not only for continued tumor growth but
also for access to the vasculature and hence for metastasis. 59

• How do growing tumors develop a blood supply?
• Tumor angiogenesis is controlled by the balance between
angiogenic factors and factors that inhibit angiogenesis. Most
human tumors do not induce angiogenesis. They remain small or
in situ for years until the angiogenic switch terminates this stage
of vascular quiescence.
• The molecular basis of the angiogenic switch involves increased
production of angiogenic factors and/or loss of angiogenesis
inhibitors. These factors may be produced directly by the tumor
cells themselves or by inflammatory cells (e.g., macrophages) or
other stromal cells associated with the tumors.
• The angiogenic switch is controlled by several physiologic
stimuli such as hypoxia. Relative lack of oxygen stimulates
production of a variety of pro-angiogenic cytokines such as
vascular endothelial growth factor (VEGF), through activation of
hypoxia-induced factor-1α (HIF1α), an oxygen-sensitive
transcription factor. HIF1α is continuously produced.
• But in normoxic settings the von Hippel-Lindau protein (VHL)
binds to HIF1α leading to ubiquitination and destruction of
HIF1α. 60

• In hypoxic conditions such as a tumor that has reached a critical
size, the lack of oxygen prevents HIF1α recognition by VHL and
it is not destroyed.
• HIF1α translocates to the nucleus and activates transcription of
its target genes such as VEGF.
• Because of these activities, VHL acts as a tumor suppressor gene
and germ-line mutations of the VHL gene are associated with
hereditary renal cell cancers, pheochromocytomas, hemangiomas
of the central nervous system, retinal angiomas, and renal cysts
(VHL syndrome).
• Both pro- and anti-angiogenic factors are regulated by many
other genes frequently mutated in cancer. For example, in normal
cells, p53 can stimulate expression of anti-angiogenic molecules,
such as thrombospondin-1, and repress expression of pro-
angiogenic molecules, such as VEGF. loss of p53 in tumor cells
not only removes the cell cycle checkpoints listed above, but also
provides a more permissive environment for angiogenesis. The
transcription of VEGF is also influenced by signals from the
RAS-MAP kinase pathway, and mutations of RAS or MYC up-
regulate the production of VEGF. 61

6. Ability to Invade and Metastasize
62

6. Ability to Invade and Metastasize
63

• The spread of tumors is a complex process involving a series of sequential
steps. This sequence of steps may be interrupted at any stage by either host
related or tumor related factors. The metastatic cascade can be subdivided
into two phases: 1) Invasion of ECM and 2) vascular dissemination and
homing of tumor cells.
• Human tissues are organized into a series of compartments separated from
each other by two types of ECM 1) basement membranes 2) Interstitial
connective tissue. Though organized differently each of these components of
ECM is composed of collagens, glycoproteins, and proteoglycans.
• The tumor cells must interact with the ECM at several stages in the
metastatic cascade. A carcinoma first must breach the underlying basement
membrane then traverse the interstitial connective tissue and ultimately gain
access to the circulation by penetrating the vascular basement membrane.
• This cycle is repeated when tumor cell emboli extravasate at a distant site.
Thus to metastasize a tumor cell must cross several different basement
membranes as well as negotiate through at least two interstitial matrices.
• Invasion of the ECM is an active process that requires four steps
1) Detachment of tumor cells from each other
2) Degradation of ECM
3) Attachment to novel ECM components
4) Migration of tumor cells
64

1) Detachment of tumor cells from each other
• The first step in the metastatic cascade is a loosening of tumor
cells. E-cadherins act as intercellular glues and their cytoplasmic
portions bind to β-catenin.
• Adjacent E-cadherin molecules keep the cells together E-cadherin
can transmit antigrowth signals by sequestering β-catenin.
• E-cadherin function is lost in almost all epithelial cancers either by
mutational inactivation of E-cadherin genes by activation of β-
catenin genes or by inappropriate expression of the SNAIL and
TWIST transcription factors, which suppress E-cadherin
expression.
65

2) Degradation of ECM
• The second step in invasion is local degradation of the basement
membrane and interstitial connective tissue.
• Tumor cells may either secrete proteolytic enzymes themselves or induce
stromal cells (e.g., fibroblasts and inflammatory cells) to elaborate
proteases. Multiple different families of proteases, such as matrix
metalloproteinases (MMPs), cathepsin D, and urokinase plasminogen
activator, have been implicated in tumor cell invasion.
• MMPs regulate tumor invasion not only by remodeling insoluble
components of the basement membrane and interstitial matrix but also by
releasing ECM-sequestered growth factors. cleavage products of
collagen and proteoglycans also have chemotactic, angiogenic, and
growth-promoting effects. For example, MMP-9 is a gelatinase that
cleaves type IV collagen of the epithelial and vascular basement
membrane and also stimulates release of VEGF from ECM-sequestered
pools.
• Benign tumors of the breast, colon, and stomach show little type IV
collagenase activity, whereas their malignant counterparts overexpress
this enzyme.
• the levels of metalloproteinase inhibitors are reduced so that the balance
is tilted greatly toward tissue degradation. overexpression of MMPs and
other proteases have been reported for many tumors.
66

3) Attachment to novel ECM components
• The third step in invasion involves changes in attachment of tumor
cells to ECM proteins.
• Normal epithelial cells have receptors such as integrins for
basement membrane laminin and collagens that are polarized at
their basal surface these receptors help to maintain the cells in a
resting differentiated state.
• Loss of adhesion in normal cells leads to induction of apoptosis.
Tumor cells are resistant to this form of cell death. The matrix itself
is modified in ways that promote invasion and metastasis. For
example, cleavage of the basement membrane proteins collagen IV
and laminin by MMP-2 or MMP-9 generates novel sites that bind to
receptors on tumor cells and stimulate migration.
67

4) Migration of tumor cells
• Locomotion is the final step of invasion propelling tumor
cells through the degraded basement membranes and zones
of matrix proteolysis.
• Migration is a complex multistep process that involves
many families of receptors and signaling proteins that
eventually impinge on the actin cytoskeleton. The
movement seems to be potentiated and directed by tumor
cell-derived cytokines such as autocrine motility factors.
cleavage products of matrix components (e.g., collagen,
laminin) and some growth factors (e.g., insulin-like growth
factors I and II) have chemotactic activity for tumor cells.
• Stromal cells also produce paracrine effectors of cell
motility such as hepatocyte growth factor/scatter factor
(HGF/SCF) which bind to receptors on tumor cells.
• Concentrations of HGF/SCF are elevated at the advancing
edges of the highly invasive brain tumor glioblastoma
multiforme supporting their role in motility.
68

 Vascular Dissemination and Homing of Tumor Cells
• In the circulation tumor cells are vulnerable to destruction by host immune cells.
• In the bloodstream, some tumor cells form emboli by aggregating and adhering to circulating
leukocytes, particularly platelets. aggregated tumor cells are thus afforded some protection from the
antitumor host effector cells.
• Most tumor cells circulate as single cells. Extravasation of free tumor cells or tumor emboli
involves adhesion to the vascular endothelium followed by egress through the basement membrane
into the organ parenchyma by mechanisms similar to those involved in invasion.
• The site of extravasation and the organ distribution of metastases generally can be predicted by the
location of the primary tumor and its vascular or lymphatic drainage.
• Many tumors metastasize to the organ that represents the first capillary bed they encounter after
entering the circulation. some tumors (e.g., lung cancers) tend to involve the adrenals with some
regularity but almost never spread to skeletal muscle. Such organ tropism may be related to the
expression of adhesion molecules by tumor cells whose ligands are expressed preferentially on the
endothelium of target organs.
• Another mechanism of site-specific homing involves chemokines and their receptors. chemokines
participate in directed movement (chemotaxis) of leukocytes and it seems that cancer cells use
similar tricks to home in on specific tissues.
• Human breast cancer cells express high levels of the chemokine receptors CXCR4 and CCR7. The
ligands for these receptors (i.e., chemokines CXCL12 and CCL21) are highly expressed only in
those organs where breast cancer cells metastasize. observation of this it is speculated that blockade
of chemokine receptors may limit metastases. After extravasation tumor cells are dependent on a
receptive stroma for growth tumors may fail to metastasize to certain target tissues because they
present a nonpermissive growth environment.
69

 Molecular Genetics of Metastasis
• Metastasis is a complex phenomenon involving a variety of steps
and pathways. It is thought that a proteins like p53 and RB play
a key role.
• Genes that function as "metastasis oncogenes" or "metastatic
suppressors" are rare. Candidates for metastasis oncogenes are
SNAIL and TWIST. which encode transcription factors whose
primary function is to promote a process called epithelial-to-
mesenchymal transition (EMT).
• In EMT carcinoma cells down-regulate certain epithelial markers
(e.g E-cadherin) and up-regulate certain mesenchymal markers
(e.g vimentin and smooth muscle actin).
• These changes are believed to favor the development of a
promigratory phenotype that is essential for metastasis.
• Loss of E-cadherin expression seems to be a key event in EMT,
and SNAIL and TWIST are transcriptional repressors that
promote EMT by down-regulating E-cadherin expression.
• EMT has been documented mainly in breast cancers; whether
this is a general phenomenon remains to be established.
70

 Genomic Instability-Enabler of Malignancy
• Six defining features of malignancy and the genetic alterations
that are responsible for the phenotypic attributes of cancer cells.
The humans literally swim in environmental agents that are
mutagenic (e.g., chemicals, radiation, sunlight) cancers are
relatively rare outcomes of these encounters.
• This state of affairs results from the ability of normal cells to
repair DNA damage. The importance of DNA repair in
maintaining the integrity of the genome is highlighted by several
inherited disorders in which genes that encode proteins involved
in DNA repair are defective.
• Individuals born with such inherited defects in DNA repair
proteins are at a greatly increased risk of developing cancer.
• Genomic instability occurs when both copies of the gene are lost
recent work has suggested that at least a subset of these genes
may promote cancer in a haploinsufficient manner.
• Defects in three types of DNA repair systems-mismatch repair,
nucleotide excision repair, and recombination repair.
71

• Genomic Instability-Enabler of Malignancy
 Hereditary Nonpolyposis Colon Cancer Syndrome (mismatch repair)
• The role of DNA repair genes in predisposition to cancer is illustrated dramatically by
hereditary nonpolyposis colon carcinoma (HNPCC) syndrome.
• This is characterized by familial carcinomas of the colon affecting predominantly the
cecum and proximal colon results from defects in genes involved in DNA mismatch
repair. When a strand of DNA is being repaired these genes act as "spell checkers."
• For example, if there is an erroneous pairing of G with T rather than the normal A with T,
the mismatch repair genes correct the defect.
• Without these "proofreaders," errors gradually accumulate in several genes, including
proto-oncogenes and cancer suppressor genes.
• Mutations in at least four mismatch repair genes have been found to underlie HNPCC.
Each affected individual inherits one defective copy of one of several DNA mismatch
repair genes and acquires the second hit in colonic epithelial cells.
• DNA repair genes behave like tumor suppressor genes in their mode of inheritance, but in
contrast to tumor suppressor genes (and oncogenes), they affect cell growth only
indirectly-by allowing mutations in other genes during the process of normal cell division.
• One of the hallmarks of patients with mismatch repair defects is microsatellite instability
(MSI). Microsatellites are tandem repeats of one to six nucleotides found throughout the
genome. In normal people, the length of these microsatellites remains constant.
• In patients with HNPCC these satellites are unstable and increase or decrease in length.
HNPCC accounts only for 2% to 4% of all colonic cancers, MSI can be detected in about
15% of sporadic cancers. The growth-regulating genes that are mutated in HNPCC
patients have not yet been fully characterized.
72

 Xeroderma Pigmentosum (nucleotide excision repair)
• Patients with another inherited disorder, xeroderma pigmentosum,
are at increased risk for the development of cancers of the skin
exposed to the ultraviolet (UV) light contained in sun rays.
• The basis of this disorder is defective DNA repair. UV light causes
cross-linking of pyrimidine residues, preventing normal DNA
replication.
• Such DNA damage is repaired by the nucleotide excision repair
system. Several proteins are involved in nucleotide excision repair,
and an inherited loss of any one can give rise to xeroderma
pigmentosum.
73

• Diseases with Defects in DNA Repair by Homologous
Recombination (recombination repair.)
• A group of autosomal recessive disorders comprising Bloom syndrome, ataxia-telangiectasia,
and Fanconi anemia is characterized by hypersensitivity to other DNA-damaging agents, such
as ionizing radiation (Bloom syndrome and ataxia-telangiectasia), or DNA cross-linking agents,
such as nitrogen mustard (Fanconi anemia).
• Their phenotype is complex and includes in addition to predisposition to cancer features such as
neural symptoms (ataxia-telangiectasia), anemia (Fanconi anemia), and developmental defects
(Bloom syndrome). The gene mutated in ataxia-telangiectasia is ATM which seems to be
important in recognizing and responding to DNA damage caused by ionizing radiation.
• Mutations in two genes, BRCA1 and BRCA2, account for 80% of cases of familial breast cancer.
In addition to breast cancer, women with BRCA1 mutations have a substantially higher risk of
epithelial ovarian cancers, and men have a slightly higher risk of prostate cancer. mutations in
the BRCA2 gene increase the risk of breast cancer in both men and women as well as cancer of
the ovary, prostate, pancreas, bile ducts, stomach, and melanocytes. cells that lack these genes
develop chromosomal breaks and severe aneuploidy. both genes seem to function, at least in
part, in the homologous recombination DNA repair pathway.
• For example, BRCA1 forms a complex with other proteins in the homologous recombination
pathway and is also linked to the ATM checkpoint pathway. BRCA2 was identified as one of
several genes mutated in Fanconi anemia and the BRCA2 protein has been shown to bind to
RAD51, a protein required for catalysis of the primary reaction of homologous recombination.
• Other tumor suppressor genes, both copies of BRCA1 and BRCA2 must be inactivated for
cancer to develop. linkage of BRCA1 and BRCA2 to familial breast cancers is established, these
genes are rarely inactivated in sporadic cases of breast cancer. In this regard, BRCA1 and
BRCA2 are different from other tumor suppressor genes, such as APC and p53, which are
inactivated in both familial and sporadic cancers.
74

CANCER TREATMENT MODALITIES
Chemotherapy is presently used in three main clinical settings:
(1) Primary induction treatment for advanced disease or for cancers
for which there are no other effective treatment approaches.
(2) Neoadjuvant treatment for patients who present with localized
disease, for whom local forms of therapy such as surgery or
radiation, or both, are inadequate by themselves,
(3) Adjuvant treatment to local methods of treatment, including
surgery, radiation therapy, or both.
75

• Primary chemotherapy refers to chemotherapy administered
as the primary treatment in patients who present with advanced
cancer for which no alternative treatment exists. The goals of
therapy are to relieve tumor related symptoms, improve overall
quality of life, and prolong time to tumor progression.
• In adults, these curable cancers include Hodgkin’s and non-
Hodgkin’s lymphoma, acute myelogenous leukemia, germ cell
cancer, and choriocarcinoma.
• The curable childhood cancers include acute lymphoblastic
leukemia, Burkitt’s lymphoma, Wilms’ tumor, and embryonal
rhabdomyosarcoma.
76

• Neoadjuvant chemotherapy patients who present with localized
cancer for which alternative local therapies such as surgery exist
but which have been shown to be less than completely effective.
• Neoadjuvant therapy is most often administered in the treatment of
anal cancer, bladder cancer, breast cancer, gastroesophageal cancer,
laryngeal cancer, locally advanced non-small cell lung cancer
(NSCLC), osteogenic sarcoma, and locally advanced rectal cancer.
• The diseases such as anal cancer, gastroesophageal cancer,
laryngeal cancer, non-small cell lung cancer(NSCLC), rectal cancer,
benefit is derived when chemotherapy is administered with
radiation therapy either concurrently or sequentially. The goal is to
reduce the size of the primary tumor so that surgical resection can
be made easier and more effective.
• In rectal cancer and laryngeal cancer, the administration of
combined modality therapy prior to surgery can result in sparing of
vital normal organs, such as the rectum or larynx. additional
chemotherapy is given for a defined period of time, usually 3–4
months, after surgery has been performed.
77

• Adjuvant chemotherapy
• Most important roles for cancer chemotherapy is as an
adjuvant to local treatment modalities such as surgery, and this
has been termed adjuvant chemotherapy. chemotherapy is
administered after surgery has been performed and the goal of
chemotherapy is to reduce the incidence of both local and
systemic recurrence and to improve the overall survival of
patients.
• Adjuvant chemotherapy is effective in prolonging both disease
free survival (DFS) and overall survival (OS) in patients with
breast cancer, colon cancer, gastric cancer, NSCLC, Wilms’
tumor, anaplastic astrocytoma and osteogenic sarcoma.
• The antihormonal agents tamoxifen, anastrozole, and letrozole
are effective in the adjuvant therapy of postmenopausal women
with early-stage breast cancer whose breast tum.
78

• BASIC PHARMACOLOGY OF CANCER
CHEMOTHERAPEUTIC DRUGS
• ALKYLATING AGENTS
• Alkylating agents exert their cytotoxic effects by alkylation of DNA
within the nucleus represents the major interaction leading to cell death..
• The MOA of drugs involves intramolecular cyclization to form an
ethyleneimonium ion that may directly or through formation of a
carbonium ion transfer an alkyl group to a cellular constituent.
• The major site of alkylation within DNA is the N7 position of guanine
other bases are also alkylated albeit to lesser degrees, including N1 and N3
of adenine, N3 of cytosine, and O6 of guanine, and phosphate atoms and
proteins associated with DNA.
• Interactions occur on a single strand or both strands of DNA through
cross-linking, as most major alkylating agents are bifunctional, with two
reactive groups. Alkylation of guanine can result in miscoding through
abnormal base pairing with thymine or in depurination by excision of
guanine residues.
• The effect leads to DNA strand breakage through scission of the sugar-
phosphate backbone of DNA. Cross-linking of DNA appears to be of
major importance to the cytotoxic action of alkylating agents and
replicating cells are most susceptible to these drugs.
81

Cyclophosphamide is one of the most widely used alkylating agents. It is
inactive in its parent form and must be activated to cytotoxic metabolites by liver
microsomal enzymes.
• The cytochrome P450 mixed function oxidase system converts
cyclophosphamide to 4-hydroxycyclophosphamide it is equilibrium with
aldophosphamide.
• The active metabolites are delivered to both tumor and normal tissue where non-
enzymatic cleavage of aldophosphamide to the cytotoxic forms—phosphoramide
mustard and acrolein—occurs. The liver appears to be protected through the
enzymatic formation of the inactive metabolites 4-ketocyclophosphamide and
carboxyphosphamide.
84

ALKYLATING AGENTS (NITROSOUREAS)
• These drugs appear to be non-cross-resistant with other alkylating
agents all require biotransformation which occurs by nonenzymatic
decomposition to metabolites with both alkylating and
carbamoylating activities.
• The nitrosoureas are highly lipid-soluble and able to cross the BBB
effective in the treatment of brain tumors.
• majority of alkylations by the nitrosoureas are on the N7 position
of guanine in DNA, the critical alkylation responsible for
cytotoxicity appears to be on the O6 position of guanine, which
leads to G-C crosslinks in DNA.
• oral administration of lomustine, peak plasma levels of metabolites
appear within 1–4 hours CNS concentrations reach 30–40% of the
activity present in the plasma. Urinary excretion appears to be the
major route of elimination from the body. One naturally occurring
sugar-containing nitrosourea streptozocin is interesting because it
has minimal bone marrow toxicity. This agent has activity in the
treatment of insulin-secreting islet cell carcinoma of the pancreas.
86

• NON-CLASSIC ALKYLATING AGENTS
• These agents include procarbazine, dacarbazine.
• Procarbazine
• Procarbazine it is derivative of a methylhydrazine.
• The MOA of procarbazine is it inhibits DNA, RNA, protein
biosynthesis.
• Oxidative metabolism of this drug by microsomal enzymes
(cytochrome P450) generates azoprocarbazine and H2O2
responsible for DNA strand scission.
• Dacarbazine is a synthetic compound that functions as an
alkylating agent metabolic activation in the liver by oxidative N-
demethylation to the monomethyl derivative.
• This metabolite spontaneously decomposes to diazomethane
generates a methyl carbonium ion that is believed to be the key
cytotoxic species.
88

• PLATINUM ANALOGS
• Three platinum analogs are used in clinical practice:
• Cisplatin, Carboplatin, and Oxaliplatin
• Cisplatin (cis-diamminedichloroplatinum [II]) is an inorganic metal complex.
• MOA of the platinum analogs is unclear cytotoxic effects in the same manner as
alkylating agents.
• It hen bindes to N7 of guanine in DNA forming inter and intrastrand cross links.
• The resulting cytotoxic lesion inhibits both DNA replication and RNA synthesis.
• Cytotoxicity can occur at any stage of cell cycle, but cells are more vulnerably to
the action of these drugs in the G1, S phases.
• The platinum analogs have been shown to bind to both cytoplasmic and nuclear
proteins may also contribute to their cytotoxic and antitumor effects.
• Carboplatin is a second-generation platinum analog.
• Oxaliplatin is a third-generation diaminocyclohexane platinum analog.
89

• ANTIMETABOLITES
• ANTIFOLATES
• Methotrexate
• Methotrexate (MTX) is a folic acid analog that
binds to the active catalytic site of
dihydrofolate reductase (DHFR). Inhibition of
the synthesis of tetrahydrofolate (THF).
Inhibition of these various metabolic processes
thereby interferes with the formation of DNA,
RNA, and key cellular proteins.
• Intracellular formation of polyglutamate
metabolites with the addition of up to 5–7
glutamate residues is critically important for
the therapeutic action of MTX and this process
is catalyzed by the enzyme folylpolyglutamate
synthase (FPGS). MTX polyglutamates are
selectively retained within cancer cells and
they display increased inhibitory effects on
enzymes involved in de novo purine nucleotide
and thymidylate biosynthesis, making them
important determinants of MTX’s cytotoxic
action.
• Pralatrexate is a 10-deaza-aminopterin
antifolate analog
90

• FLUOROPYRIMIDINES
• 5-Fluorouracil
• 5-Fluorouracil (5-FU) is inactive
form.
• Its metabolites 5-fluoro-2′-
deoxyuridine-5′-monophosphate
(FdUMP) forms a covalently bound
ternary complex with the enzyme
thymidylate synthase and the reduced
folate 5,10-methylenetetrahydrofolate,
a reaction critical for the de novo
synthesis of thymidylate.
• This results in inhibition of DNA
synthesis through “thymineless
death.” 5-FU is converted to 5-
fluorouridine-5′- triphosphate (FUTP)
which is then incorporated into RNA,
where it interferes with RNA
processing and mRNA translation.
91

• Capecitabine
• Capecitabine is a
fluoropyrimidine carbamate
prodrug.
• Metabolism in the liver by the
enzyme carboxylesterase to an
intermediate (5′-dFCR) 5′-
deoxy-5-fluorocytidine.
metabolite is converted to (5′-
dFUR) 5′-deoxy-5-fluorouridine
by the enzyme cytidine
deaminase.
• The 5′-deoxy-5- fluorouridine
metabolite is finally hydrolyzed
by thymidine phosphorylase to
5-FU directly in the tumor.
92

• DEOXYCYTIDINE ANALOGS
• Cytarabine is an S phase-specific.
• Gemcitabine is a fluorine-substituted deoxycytidine analog that is
phosphorylated by the enzyme deoxycytidine kinase to the
monophosphate form and by other nucleoside kinases to the
diphosphate and triphosphate nucleotide forms.
• The antitumor effect is result from several mechanisms: inhibition of
ribonucleotide reductase by gemcitabine diphosphate reduces the
level of deoxyribonucleoside triphosphates required for DNA
synthesis. Inhibition by gemcitabine triphosphate of DNA
polymerase-α and DNA polymerase-β thereby resulting in blockade
of DNA synthesis and DNA repair and incorporation of gemcitabine
triphosphate into DNA leading to inhibition of DNA synthesis and
function.
93

• PURINE ANTAGONISTS
• 6-Thiopurines
• 6-Mercaptopurine (6-MP) was the first of the thiopurine analogs.
• 6-MP is inactive in its parent form and metabolized by hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) to form the monophosphate nucleotide 6-
thioinosinic acid which in turn inhibits several enzymes of de novo purine
nucleotide synthesis.
• The monophosphate nucleotide 6-thioinosinic acid form is eventually metabolized
to the triphosphate form which can then be incorporated into both RNA and DNA.
• 6-Thioguanine (6-TG) also inhibits several enzymes in the de novo purine
nucleotide biosynthetic pathway.
• 6-MP is converted to an inactive metabolite (6-thiouric acid) by an oxidation
reaction catalyzed by xanthine oxidase whereas 6-TG undergoes deamination.
• This is an important issue because the purine analog allopurinol a potent xanthine
oxidase inhibitor is frequently used as a supportive care measure in the treatment
of acute leukemias to prevent the development of hyperuricemia that often occurs
with tumor cell lysis. Because allopurinol inhibits xanthine oxidase, simultaneous
therapy with allopurinol and 6-MP would result in increased levels of 6-MP
thereby leading to excessive.
95

• Fludarabine
• Fludarabine phosphate is rapidly dephosphorylated to 2-fluoro-
arabinofuranosyladenosine and then phosphorylated intracellularly
by deoxycytidine kinase to the triphosphate.
• The triphosphate metabolite interferes with the processes of DNA
synthesis and DNA repair through inhibition of DNA polymerase-α
and DNA polymerase-β.
• The diphosphate metabolite of fludarabine inhibits ribonucleotide
reductase, leading to inhibition of essential deoxyribonucleotide
triphosphates.
• Cladribine
• Cladribine (2-chlorodeoxyadenosine) is a purine nucleoside analog.
• Inactive in its parent form initially phosphorylated by deoxycytidine
kinase to monophosphate form and eventually metabolized to the
triphosphate form, which can then be incorporated into DNA.
• The triphosphate metabolite can also interfere with DNA synthesis
and DNA repair by inhibiting DNA polymerase-α and DNA
polymerase-β. 97

• NATURAL PRODUCT CANCER
CHEMOTHERAPY DRUGS
• VINCAALKALOIDS
• Vinblastine, Vincristine, Vinorelbine
• Vinca alkaloid derived from the
periwinkle plant Vinca rosea .
• Its MOA involves inhibition of tubulin
polymerization.
• Disrupts assembly of microtubules.
• Inhibitory effect results in mitotic arrest in
metaphase bringing cell division to a halt
which then leads to cell death.
98

• TAXANES & RELATED DRUGS
• Paclitaxel is an alkaloid ester derived
from the Pacific yew (Taxus brevifolia)
and the European yew (Taxus baccata ).
• Docetaxel is a semisynthetic taxane
derived from the European yew tree.
• Cabazitaxel is a semisynthetic taxane
produced from a precursor extracted from
the yew tree.
• The drug functions as a mitotic spindle
poison through high-affinity binding to
microtubules with enhancement of tubulin
polymerization.
• The promotion of microtubule assembly
by these drugs occurs in the absence of
microtubule-associated proteins and
guanosine triphosphate and results in
inhibition of mitosis and cell division.
• It is metabolized extensively by the liver
CYP450 system.
99

• EPIPODOPHYLLOTOXINS
• Etoposide is a semisynthetic derivative of Podophyllotoxin.
• Extracted from the mayapple root (Podophyllum peltatum).
• The main site of action is inhibition of the DNA enzyme
topoisomerase II.
100

• CAMPTOTHECINS
• Topotecan and Irinotecan are the two camptothecin analogs.
• The camptothecins are natural products derived from the
Camptotheca acuminata tree originally found in China; they inhibit
the activity of topoisomerase I, the key enzyme responsible for
cutting and religating single DNA strands.
• Inhibition of this enzyme results in DNA damage.
101

ANTHRACYCLINES
Doxorubicin, Daunorubicin, Idarubicin,
Epirubicin, Mitoxantrone
The anthracycline antibiotics isolated from
Streptomyces peucetius var caesius, are among
the most widely used cytotoxic anticancer drugs.
The anthracyclines exert their cytotoxic action
through four major mechanisms:
(1) Inhibition of topoisomerase II
(2) High-affinity binding to DNA through
intercalation, with consequent blockade of the
synthesis of DNA and RNA, and DNA strand
scission
(3) Generation of semiquinone free radicals and
oxygen free radicals through an iron-
dependent, enzyme-mediated reductive
process;
(4) Binding to cellular membranes to alter fluidity
and ion transport.anthracyclines are
administered via the intravenous route.
102

ANTITUMOR ANTIBIOTICS
Mitomycin (mitomycin C) is an antibiotic
isolated from streptomyces caespitosus. It
undergoes metabolic activation through an
enzyme-mediated reduction to generate an
alkylating agent that crosslinks DNA.
Bleomycin is a small peptide that contains a
DNA-binding region and an iron-binding
domain at opposite ends of the molecule.
It acts by binding to DNA, which results in
single- and doublestrand breaks following
free radical formation, and inhibition of
DNA biosynthesis.
The fragmentation of DNA is due to oxidation
of a DNA bleomycin-Fe(II) complex and
leads to chromosomal aberrations.
103

Reference:-
• Robbins basic Pathology 8th edition pp no; 173-224.
• Principles of Pharmacology the Pathophysiologic Basis
of Drug therapy David E. Golan, 3RD Pp no: 699-715 and
4th edition pp no: 750-764.
• Text book of Pathology 7th edition by Harsh mohan
pp no 184-250.
105

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