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In Carcinogenesis
Dolores V. Viliran, MD
CANCER
Cancer is an overgrowth of cells bearing
cumulative genetic injuries that confer growth
advantage over the normal cells [Nowell’s
Law]
Normal
cell
Nucleus
Few
Blood vessel mitoses
Abnormal
heterogeneous cells
• Autonomy
• Anaplasia
• Metastasis
CHARACTERISTICS OF CANCER
Clonality
• Cancer is a genetic disease at the cellular level.
• Genetic mutations play a critical role in
pathogenesis of cancer.
• Consequences of genetic instability:
– Phenotypic heterogeneity
– Tumor progression
• Proto-oncogenes and oncogenes
• Dominant mutations = mutation resulting from
conversion of protooncogenes to oncogenes
• Recessive mutations = mutation resulting from
damage or loss of tumor suppressor gene.
Cancer Genetics
• Tumors arise as clones from a single
cell. At the cellular level, cancer is a
genetic disease.
• The development of the malignant clone
is due to mutations in DNA due to:
– Random replication errors
– Exposure to carcinogens
– Faulty DNA repair process
Evidence that Mutations Cause
Cancer
• Recurring sites of chromosome change are
observed in cancers at sites of genes
involved in cellular growth control.
• Most carcinogens are mutagens.
• Defects in DNA repair systems increase
the possibility of cancer.
CHARACTERISTICS OF CANCER
Autonomy
• Cancer cells are able to proliferate despite
regulatory influences.
• Unrestricted proliferation results in tumor
formation.
• Mechanisms:
– Growth factor secretion
– Increased number of cell receptors
– Independent activation of key biochemical
process
• Proliferation depends on the cell cycle.
AUTONOMY
• Brought about by mutations in the cell’s
genetic apparatus
• Most common in tissues with rapid
turnover, especially:
- those exposed to environmental agents
- those whose proliferation is hormone-
dependent
• Proliferation is dependent on the cell
regeneration cycle
The Cell Cycle
DEATH
G0
DIFFERENTIATION
G2/M checkpoint
Mitosis
M
DNA content = 2n
DNA content = 4n
G2 G1
S G1/S checkpoint
DNA synthesis
CYCLIN, CDK,CDKI:
PHOSPHORYLATION
Cell Cycle Regulation
• Process assures that cell accurately
duplicates its contents.
• Important checkpoints are present at
G1 and G2 and are regulated by
protein kinases called cyclins (cdk).
• Checkpoints determine whether the
cell proceeds to next phase of the
cycle.
G2/M Checkpoint
• Regulated by the cyclin B/cdc2
(mitosis promoting factor or MPF).
• Activity of this cyclin with its
substrate results in:
– Chromosome condensation
– Nuclear membrane breakdown
– Spindle formation
G1/S Checkpoint
• Area most often disrupted in cancer.
• Mechanism of regulation is complex
and involves the phosphorylation of
the Rb gene. This results in:
– Activation of several genes needed for S
phase progression.
– Promotes differentiation through
association with transcription factors.
Rb Gene Activation
Cyclin Regulators
• Regulated by cdk inhibitors (cdki).
• May be induced by growth inhibitors
and inhibited by positive growth
factors.
• Genetic alterations in cdki occur with
high frequency in some cancers.
Cyclin Regulators
• p 21: inhibits cell cycle progression and
permits DNA repair to take place.
• P53: “the guardian of the genome”
– In the presence of DNA damage, influences
transcription to either:
• Halt cell cycle progression to facilitate DNA repair.
• In cases of severe DNA damage, activates apoptosis.
– Mutations in p53 are the most common genetic
alterations found in human cancer.
CHARACTERISTICS OF
CANCER: Anaplasia
• Loss of differentiated function
resulting to bizarre-looking cells
• Large nuclei, prominent nucleoli,
increased chromatin
• Increased and/or abnormal mitosis
• Aneuploidy
• Partial or complete loss of normal
architecture
Invasion and Metastasis
• The defining characteristic of a
malignancy.
• Invasion: active translocation of
neoplastic cells across tissue
barriers.
• Critical pathologic point: local invasion
and neovascularization. These events
may occur before clinical detection.
ATTRIBUTES OF CANCER
Metastasis
Two basic steps:
Destruction of the BM
Attachment to the laminin of distant BM
• Local proteolysis
Mutation
1 - Oncogenes
2 - Tumor Suppressor genes
3 - Mutator genes
Cancer Genes
• Proto-oncogenes – normally promote normal
cell growth; mutations convert them to
oncogenes.
• Tumor suppressor genes – normally restrain
cell growth; loss of function results in
unregulated growth.
• Mutator or DNA repair genes – when
faulty, result in an accumulated rate of
mutations.
ONCOGENE FAMILY
+ oncogenes
Oncogenes
promote cell proliferation
dominant & highly conserved
types: viral oncogenes [v-oncs]
cellular oncogenes [c-oncs]
Proto-oncogene ⇒ “Mutation” ⇒ Oncogene
ONCOGENE FAMILY
Classification of Oncogenes
2. Gene Amplification
Double minutes
HSRs
Homogenously
Staining regions Normal copy Multiple copies
ONCOGENE FAMILY
3. Gene Translocation
3. Gene Translocation
promoter
Viral promoter
TUMOR SUPPRESSOR GENE
FAMILY
TS Genes
inhibit growth and multiplication of mutated cells
prevent neoplastic transformation
recessive & highly conserved
Classification of TS genes
A. Cell Adhesion Molecules
APC, DCC
pRb
location: 17p13.1
product: p53 protein [53 KDa]
function: induces DNA repair or apoptosis
mutation: point mutation > deletion
results to: loss of function & extended lifespan of p53
Clinical conditions: carcinomas, Li Fraumeni
Syndrome
p53 inhibited by: E1B, HPV E6, mdm2
TUMOR SUPPRESSOR GENE
FAMILY
p53 protein
p53 in action
MUTATOR GENE FAMILY
Mutator Genes
involved in ensuring the fidelity of replication
function: checks for & corrects mismatched pairs
mutation ⇒ inefficient repair & replication leading
increased propensity of oncogenes and tumor
suppressor genes to undergo mutation
first described in E coli [Mut-HSL system]
Fischel, et al = Human homologs
leads to the formation of Microsatellite Instability
[MIN+]
In summary …..
MUTATOR GENES
ONCOGENES TS GENES
Re-cap of Molecular
Carcinogenesis
Proto-oncogene Gain-of-function
TS gene Loss-of-function CANCER
Sexual promiscuity
Multiple partners
Unsafe Sex Cervical Cancer
Human Papillomavirus
Multifactorial Factors
Oral Cavity Cancer
Tobacco + Alcohol
Esophageal Cancer
Tobacco + Asbestos
Tobacco + mining Respiratory
Tract Cancer
Tobacco + uranium + Lung Cancer
radium
CARCINOGEN METABOLISM
I. Chemical Carcinogens
II. Physical Carcinogens
III. Viral Agents
Environmental ?
factors
CHEMICAL CARCINOGENESIS
Stages:
Initiation - primary exposure
Promotion - transformation
Frank Cancer
CHEMICAL CARCINOGENESIS
Initiation
normal cells are exposed to a carcinogen
not enough to cause malignant transformation
requires one round of cell division
normal cells are exposed to a carcinogen
1. Direct-acting carcinogens
2. Indirect-acting carcinogens
Cytochrome Ultimate
procarcinogen
P450 carcinogen
CHEMICAL CARCINOGENESIS
Promotion
initiated cells are exposed to promoters
promoters are not carcinogens !
properties of promoters reversible
dose-dependent
time-dependent
Types of Carcinogens …...
1. Direct carcinogens
2. Procarcinogens ⇒ Ultimate carcinogens
CHEMICAL CARCINOGENESIS
Direct-acting Procarcinogens
Carcinogens
PAHs
cyclophosphamide Aromatic amines & Azo dyes
chlorambucil Aflatoxin B1
busulfan Nitrosamine & Amides
melphalan Asbestos
Vinyl chloride
Promoters
saccharine & cyclamates Chromium, nickel, other metals
Estrogen Arsenic
Diesthystilbestrol [DES]
Physical Carcinogenesis
• Radiation-induced mutation in the
host cell
• Transmits irreversible changes in
gene expression to cell progeny
Sources of Potentially
Carcinogenic Radiation
• Sunlight
• Artificial sources of UV light
• X-rays
• Radio-chemicals
• Nuclear fission
PHYSICAL CARCINOGENESIS
Ultraviolet Rays
Ultraviolet Rays
UV-C ⇒ filtered by ozone
UV-B
Ionizing Radiation
includes electromagnetic rays & particulate matter
mechanism: ↑ free radicals & mutations
pathology: leukemias > thyroid ca > lung & breast ca
resistant tissues: bone, skin and the GIT
PRE-IRRADIATION POST-IRRADIATION
Viral Carcinogenesis
• Viral carcinogens are classified into
RNA and DNA viruses.
Oncogene
Oncogene Viral RNA Oncogene
REVERSE TRANSCRIPTASE protein
Viral DNA
NUCLEUS INSERTION
DNA
CELL
MEMBRANE CYTOPLASM
Viruses Associated With The
Development Of Human Neoplasia
VIRUSES NEOPLASMS
DNA VIRUSES
Human papilloma virus Cervical Ca, warts, ano-
genital carcinoma
Herpes simplex virus II Cervical carcinoma
Epstein-Barr virus NPCa, African Burkitt’s
Herpes simplex virus 8 Kaposi’s sarcoma
Hepatitis B virus Hepatocellular Ca
Herpes simplex virus 6 Certain B cell
(HBLV) lymphomas
Viruses Associated With The
Development Of Human Neoplasia
VIRUSES NEOPLASMS
RNA VIRUSES
Human T-cell leukemia virus I Some T-cell leukemia,
lymphoma
Human T-cell leukemia virus II Some cases of hairy
cell leukemia
Human immunodeficiency virus I Lymphoma; Kaposi’s
sarcoma
VIRAL AGENTS: DNA viruses
E2 is not expressed
Over-expression of E6 & E7
VIRAL AGENTS: DNA viruses
a retrovirus
tropism: CD4+ cells
mechanism: Tax protein
G2/M checkpoint
Mitosis
M
DNA content = 2n
DNA content = 4n
G2 G1
S G1/S checkpoint
DNA synthesis
Cell Cycle Regulation
• Process assures that cell accurately
duplicates its contents.
• Important checkpoints are present at G1
and G2 and are regulated by proteins
Cyclins and Cyclin-dependent Kinases
(CDKs).
• Checkpoints determine whether the cell
proceeds to next phase of the cycle.
Cyclins and Cyclin-dependent
Kinases (CDKs)
• CYCLINS – activate protein kinases
• CDKs – protein enzymes which
selectively phosphorylate specific
serine/threonine residues in their
substrates
• Dimeric complex with
catalytic subunit (CDK 1-9)
regulatory subunit (Cyclin A-H,T)
G2/M Checkpoint
• Regulated by the cyclin B/cdc2 (mitosis promoting
factor or MPF).
• Regulated mainly by intracellular signal
(Completion of DNA Synthesis)
• MPF is activated by dephosphorylation by cdc25
• Cyclin B is degraded by Anaphase Promoting
Complex (APC)
• Role of G2/M checkpoint: to prevent mitosis when
DNA is damaged and not yet repaired
CYCLIN, CDK,CDKI:
PHOSPHORYLATION
G1/S Checkpoint
• Area most often disrupted in cancer.
• Mechanism of regulation is complex and
involves the phosphorylation of the Rb
gene.
• Regulated by extracellular signals (e.g. GF)
• “R” point (restriction)- point late in G1
beyond which cell cycle progression
becomes independent from external GF
• Regulated mainly by CDK4/cyclin D
Rb Gene Activation
Cyclin Regulators- CDK
Inhibitors
• CDK inhibitors – inhibit the activity of CDK-cyclin
complex
• Two Groups:
1) INK4 family – p15 16 18 19
2) CIP-KIP family – p21 p27
Actions:
P15- change response to anti-mitogenic agents
P16- inhibits CDK4/cyclin D
P19- induces p53 stabilization
P21-induces cell cycle arrest via activation by p53
P27- inhibits CDK2/cyclin E
Cyclin Regulators
• p 21: activated by p53 inhibiting cell cycle
progression and permitting DNA repair to
take place.
• P53: “the guardian of the genome”
– In the presence of DNA damage, influences
transcription to either:
• Halt cell cycle progression to facilitate DNA repair.
• In cases of severe DNA damage, activates apoptosis.
– Mutations in p53 are the most common genetic
alterations found in human cancer.
p53 in action
CELL-CYC LE
PR OG RES SION
Clinical Significance
Oncogenic alterations in cell cycle regulators:
• Loss of p53 and pRB function as tumor
suppressors
• Increased expression of Cyclin D1(Mantle Cell
Lymphoma)
• CDK4 amplification in sarcomas, glioma
• Mutations in p16-binding domain of CDK4(Familial
Melanoma)
• Inactivation of INK4
• Alterations in Cyclin D1,p16
• Decreased levels of p27 (Breast Ca)
• Over expression of cdc25
Therapeutic Implications
Approaches using Inhibitors of CDKs
as therapeutic agents
• Small molecules
• Protein therapy
• Antisense
• Gene therapy
Most cytotoxic agents block the cell
cycle in the S/G2/M phases
DNA REPAIR PATHWAYS
• Cancer as “Malady of Genes”
• Defects in the maintenance of genome
stability
• Repair Mechanisms:
4. Mismatch excision repair
5. Base excision repair
6. Nucleotide excision repair
7. Double strand base repair
DNA REPAIR PATHWAYS
Clinical Significance
HNPCC – mutations in genes involved in DNA
repair pathways (MSH1 MSH2)
• Somatic defects in repeated DNA elements
leading to Microsatellite instability (MSI)
• Inactivation of TGF-β (tumor suppressor)
• Inactivation of BAX gene
IMM OR TALI ZATION
Telomeres and Telomerase
Telomeres- specialized structures at
chromosome ends generated and
maintained by telomerase
Telomerase- ribonucleoprotein enzyme which
preserves the integrity of telomeres
* key component in immortalization of
cancer cells
Telomere length- represents a molecular
clock that determines the life span of the
cell
Telomeres and Telomerase
Clinical Significance
• Most normal adult tissues have NO telomerase
activity
• Telomerase activity is present in 90% of tumors
Therapeutic Implication
hTERT- protein identified to be catalytic subunit of
telomerase
• limiting component of telomerase activity
• can be a target for small molecule inhibitor
APOPTOSIS
• APOPTOSIS – programmed cell death
• Important in:
3. Steady-state kinetics of normal tissues
4. Focal deletion of cells during normal
embryonic development
5. Seen after chemotherapy and radiation
* Balance between proliferation and apoptosis
is critical in determining growth or
regression
Components of Apoptotic
Pathway
1) CASPASES (Cysteine-containing aspartate-
specific proteases)
• Initiator Caspases – activated in response to cell
death signal
• Executioner or Effector Caspases- progress the
death signal activating cascade resulting to DNA
fragmentation and cell death
Caspase prodomains – DED CARD
Death ligands – TNF-α , Fas , TRAIL
Survival Signals – NFκβ
Components of Apoptotic
Pathway
2) CYTOCHROME C – component of mitochondria
released in response to apoptotic signals
3) BCL-2 Family of Proteins- located upstream in the
pathway
• Provides pivotal decisional checkpoint in the fate
of the cell after a death stimulus
• Contains BH1-BH4 domains necessary for
interaction
• Anti-apoptotic – BCL-2 BCL-xL
• Pro-apoptotic – BAX BAD BAK BID
APOPTOTIC PATHWAYS
1) FAS-mediated apoptosis
• FAS – cell surface receptor of TNF family
which binds to FAS-L
• Eliminates unwanted activated T cells
• Pathway for cytotoxic-mediated signaling
2) P53-mediated apoptosis
• important after chemotherapy and radiation
• Induction of BAX and downregulation of BCL-2
• Induced expression of FAS and DR5
Clinical Significance
• Over expression of BCL-2 as a prognostic
indicator
• Mutations of BAX in GI Ca and leukemias
• P53 provides a link between cell
proliferation and apoptosis
• Cell survival signals: NFκβ BCL-2
• P53 mutations confer chemoresistance
EVA DING A POPTOS IS
Therapeutic Implications
• Antisense oligonucleotide against
BCL-2 in the treatment of lymphoma
• BCL-2 antisense as chemosensitizing
agent in solid tumors
• TRAIL ( TNF-related apoptosis
inducing ligand) to induce apoptosis
ANGIOGENESIS
• Formation of new blood vessels from
existing vascular bed
• Carried out by endothelial cells (EC) and
extra cellular matrix (ECM)
• Regulated by angiogenic factors (inducers
and inhibitors)
* A tumor is unable to grow larger than 1
mm3 w/o developing a new blood supply
Components of Angiogenesis
1) ENDOTHELIAL CELLS
• Fenestrated
• Increased cell adhesion molecules
( E-selectin)
• Increased integrins αγβ3 essential
for viability during growth
• Activated ECs release: bFGF PDGF
IGF-1
Components of Angiogenesis
2) INDUCERS OF ANGIOGENESIS
• VEGF – main inducer
• TGF- β
• TNF-α low concentration - inducer
high concentration -
inhibitor
• PDGF/thymidine phosphorylase
• TGF-α
• EGF
• IL-8
Components of Angiogenesis
3) CELL ADHESION MOLECULES (CAM)
• Mediate cell-cell adhesion processes
• Selectins
• IG Supergene family- ICAM VCAM
• Cadherins
• Integrins- vitronectin receptor
4) PROTEASES
• Degrade ECM to provide suitable
environment for EC migration thru adjacent
stroma Ex: Metalloproteinases (MMP)
Components of Angiogenesis
1) ANGIOGENESIS INHIBITORS
• Interferon
• TSP-1
• Angiostatin
• Endostatin
• Vasostatin
CLINICAL SIGNIFICANCE:
Tumor angiogenesis switch is triggered as
a result of shift in the balance of
stimulators to inhibitors
ANG IOG ENES IS
Therapeutic Implications
• Metalloproteinase inhibitors (MMPI) –
block the degradation of basement
membrane
• Inhibitors of endothelial function-
thalidomide, TNP 470,endostatin
• Anti-angiogenic factors – tyrosine
kinase inhibitors of VEGF bFGF PDGF
• Interferon – angiogenic inhibitor
• COX-2 inhibitor – thromboxane A2 as
critical intermediary of angiogenesis
INVASION AND METASTASIS
Invasion and Metastasis
• The defining characteristic of a
malignancy.
• Invasion: active translocation of
neoplastic cells across tissue
barriers.
• Critical pathologic point: local invasion
and neovascularization. These events
may occur before clinical detection.
PROCESS OF METASTASIS
Triad of Invasion
• Adhesion with the basement
membrane.
• Local proteolysis
• Mobility and ability to translocate
through rents in body’s structural
barriers.
ADHESION
• De-regulated function of CAM (E-cadherin)
• Changes in catenin expression leads to loss of
cadherin function
• Integrin over expression in naturally
occurring cancers
• Downregulation of integrin in more advanced
stages of cancer
• Upregulation of ICAM-1 which enhances
extravasation
• Adhesion molecules on EC: E-selectin,VCAM
ICAM
LOCAL PROTEOLYSIS
• Degradation of basement membrane
to traverse barriers
• Carried out by:
• Serine proteases -uPA elastase
plasmin cathepsin G
• Cysteine proteases- cathepsin B L
• Aspartate proteases – cathepsin D
• Matrix metalloproteinases-
gelatinases interstitial collagenases
stromelysins matrilysins
MOTILITY
• Tumor cells can move randomly or
directionally toward attractants
• Modulators of motility
GF, hyaluronases, components of ECM,
tumor-secreted factors, host-derived
factors
THERAPEUTIC IMPLICATIONS:
MMPI and monoclonal antibodies
against integrin
METAS TAS IS AND
INV AS ION
Which of the following is TRUE
of carcinogenesis?
A. Carcinogenesis occurs as a result of
genetic mutation secondary to physical and
chemical agents only
B. The ultimate carcinogens are usually
electrophiles which can readily attack NA
C. The most common base involved in
mutagenesis is adenine
D. Tumor suppressor gene is transformed to
oncogene
Which of the following is TRUE
of carcinogenesis?
• Carcinogenesis occurs as a result of
genetic mutation secondary to physical and
chemical agents only
• The ultimate carcinogens are usually
electrophiles which can readily attack
NA
• The most common base involved in
mutagenesis is adenine
• Tumor suppressor gene is transformed to
Tumor p53 suppressor protein is
reffered to as guardian of the
genome bec. it:
A. Enhances the survival of tissues
B. Allows apoptosis to occur on seriously
damaged cells
C. Plays a key role in G2 checkpoint control
D. Arrests the cell cycle at Go phase
Tumor p53 suppressor protein is
reffered to as guardian of the
genome bec. it:
• Enhances the survival of tissues
• Allows apoptosis to occur on seriously
damaged cells
• Plays a key role in G2 checkpoint control
• Arrests the cell cycle at Go phase
TRUE statements about
oncogenes, EXCEPT:
A. They positively affect cell proliferation
B. Single mutant allele is enough to cause
phenotypic
C. They are mutant protooncegenes
D. Mutation involves a loss in function
TRUE statements about
oncogenes, EXCEPT:
• They positively affect cell proliferation
• Single mutant allele is enough to cause
phenotypic
• They are mutant protooncegenes
• Mutation involves a loss in function
This is not a characteristics of
cancer
A. Loss of contact inhibition
B. Uncontrolled proliferation
C. Gain in function of mutator gene
D. Loss of differentiated function
This is not a characteristics of
cancer
• Loss of contact inhibition
• Uncontrolled proliferation
• Gain in function of mutator gene
• Loss of differentiated function
TRUE statements about RAS
oncogene activation except:
A. It involves a point mutation in codon 12
B. The mutated RAS results to increased
GTPase activity
C. The mutated gene codes for valine instead
of glycine
D. It is over-expressed in bladder cancer
TRUE statements about RAS
oncogene activation except:
• It involves a point mutation in codon 12
• The mutated RAS results to increased
GTPase activity
• The mutated gene codes for valine instead
of glycine
• It is over-expressed in bladder cancer
A biochemical change found in
fast growing tumor cells:
A. Increased catabolism of nucleobases and
nucleotides
B. Inappropriate synthesis of certain growth
factors and hormones
C. An adult pattern of isozymes
D. Markedly decreased glycolysis
A biochemical change found in
fast growing tumor cells:
• Increased catabolism of nucleobases and
nucleotides
• Inappropriate synthesis of certain
growth factors and hormones
• An adult pattern of isozymes
• Markedly decreased glycolysis
Any Questions ?