Nicnas carcinogenesis8copy

Chemical Carcinogenesis
R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT

Carcinogenesis
Definitions

Classical Concepts

Modern Molecular Concepts and Markers

Assays and Measurement

Toxicological Thresholds and Risk Assessment

Learning Objectives

 Understand the key definitions pertaining to
carcinogenesis;

 Understand the classical concepts of chemical
carcinogenesis;

 Understand the MAJOR molecular themes that underpin
carcinogenesis;

 Understand the currently available types of
carcinogenesis assays.

Definitions

 Tumor:
 Classically the term has been applied to any space
occupying lesion; particularly if the lesion is of inflammatory
origin;

 In modern times, tumor has become synonymous with
neoplasia;

 derived from the Latin word for "swelling" – tumor;

 In the Commonwealth the spelling "tumor" is commonly
used, whereas in the U.S. it is usually spelled "tumor”;

A tumor = a space occupying lesion

Definitions

 Neoplasia:
 Literally means “new growth”;
 A neoplasm is an abnormal mass of tissue, the growth of
which exceeds and is uncoordinated with that of the normal
tissues and persists in the same excessive manner after
cessation of the stimuli that evoked the change
 The entire population of cells within a neoplasm (with rare
exceptions) derive from a single transformed cell i.e. they
are clonal;

Definitions

 Neoplasia:
 All neoplasms have 2 major tissue components:
 Clonal neoplastic cells that constitute the parenchyma of
the neoplasm;
 A reactive stroma made up of connective tissue, blood
vessels (angiogenesis), and an inflammatory cellular
infiltrate (with variable numbers of macrophages and
lymphocytes)
 Tumor growth and evolution is critically dependent upon its
stroma! The stroma affects the neoplastic cells and the
neoplastic cells affect the stroma

Definitions

 Neoplasia:
 In some neoplasm types there is an abundant fibrous stroma;
 The laying down of a collagenous stroma in a neoplasm is
called desmoplasia;
 Desmoplastic tumors containing abundant collagenous
stroma are referred to as scirrhous;
 Neoplasms commonly contain areas of necrosis: thought
primarily to be due to the inability of blood/nutrient supply to
match the growth needs of the neoplastic cells (i.e.
insufficient angiogenesis);
 Some neoplasms (particularly in the skin) may have zones of
bacterial infection;

A scirrhous hepatocellular carcinoma displaying desmoplasia

Definitions

 Benign:
 In general, the suffix “oma” is used to designate a benign
neoplasm, particularly if the neoplasm is of messenchymal
origin e.g. adenoma = a benign neoplasm of glandular
origin (may or may not form glandular structures); fibroma =
a benign neoplasm of fibrous tissue;
 Benign tumors of epithelial origin include: papillomas,
cystomas etc.;

Definitions

 Features of benign neoplasms
 In general the neoplastic cells are well differentiated and
resemble those of their tissue of origin i.e. they do not display
anaplasia, pleomorphism or high mitotic rates;
 Not locally invasive e.g. generally do not spread through
basement membranes of epithelia;
 Are not metastatic i.e. do not spread to distant sites;
 Typically encapsulated within fibrous tissue;
 Produces damage to local structures primarily by
compression or distortion of the tissue;

Benign Phyllodes neoplasm of breast: note how well the
neoplastic cells appear to be organized, the lack of
local invasiveness and the fibrous capsule (this is a
fibroepithelial neoplasm; note the desmoplasia)

Just in case you thought that benign neoplasia is not destructive
and damaging!

Definitions

 Features of malignancy:
 Poor differentiation = differentiation refers to the extent to
which neoplastic cells resemble their corresponding normal
parenchymal cells both morphologically and functionally. In
general, benign neoplasms are well-differentiated where as
malignant neoplasms tend to be poorly differentiated;

Gleason Grade I prostatic adenocarcinoma:
Consists of single and separate rather uniform acini with little
intervening stroma and all acini are well demarcated from the
surrounding stroma.

Gleason Grade V prostatic adenocarcinoma
solid tumor with little gland formation. The tumor shows
poorly differentiated single cells in an infiltrative or sheet-like
pattern.

Definitions

 Anaplasia = malignant neoplasm consisting of poorly
differentiated cells. Anaplasia literally means to form
backwards i.e. a more primitive level of cell differentiation;

 However, anaplastic cells DO NOT represent reversion of an
already differentiated cell to a more primitive type.
Anaplastic cells derive from the abnormal differentiation
and maturation of tissue stem cells.

Definitions
 The key features of anaplasia are:
 Pleomorphism = marked variation in cell size and shape
within the same neoplasm;
 Hyperchromasia = nuclei contain abundant chromatin
and are thus dark staining;
 Abnormal nuclear morphology = large &/or increased
number of nucleoli; large nuclei (nuclear to cytoplasm
ratio up to 1:1);
 Excessive number of mitoses (high mitotic index);
 Loss of cell polarity = cell orientation is markedly
disturbed; sheets or masses or cells grow in an anarchic,
disorganized manner;
 Multinucleate or giant cells

Classical anaplasia: can you spot all the features?

Definitions
 Metaplasia = the replacement of one mature
differentiated cell type with another mature
differentiated cell type;

 Metaplasia almost always is associated with tissue damage,
repair and regeneration;

 Typically involves a change to a cell type that is more suited
to a change in environmental conditions;

 Metaplasia not directly considered carcinogenic.

Definitions
 Dysplasia = disordered growth;

 Dysplasia is most commonly applied to disordered epithelia;

 Dysplasia generally consists of an expansion of immature
cells, with a corresponding decrease in the number and
location of mature cell;

 Dysplasia can be adaptive (e.g. tissue repair processes) or
indicative of an early neoplastic process.

Definitions
 Dysplasia = disordered growth;

 The term dysplasia is typically used when the cellular
abnormality is restricted to the originating tissue, as in the
case of an early, in-situ neoplasm;

 Dysplasia may mean that cell maturation and differentiation
are delayed;

 Dysplasia does not necessarily mean neoplasia or
progression to neoplasia

Definitions
 Dysplasia is characterized by four major pathological
microscopic changes:

 Cellular pleomorphism;

 Anisocytosis (cells of unequal size);

 Poikilocytosis (abnormally shaped cells);

 Nuclear hyperchromatism;

 Presence of mitotic figures (an unusual number of cells
which are currently dividing); however mitotic figures are of
normal confirmation.

Definitions
 Loss of contact inhibition of cell growth. Contact inhibition is
The cessation of cellular growth and division due to physical
contact with other cells. The ability of malignant neoplastic
cells to keep growing and replicating despite contact with
other cells or structures is a classical feature of malignancy;

 Cells that lack contact inhibition will “pile up on each other”
during culture.

Definitions
 Local invasiveness:

 Almost all benign neoplasms develop as a cohesive
expansile mass that remains localized at the site of origin.
Typically benign neoplasms are encapsulated i.e. there is
a clearly defined “cleavage plane”

Benign dermal neoplasm with a clean cleavage line

Definitions
 Local invasiveness

 Malignant neoplasms are invasive (i.e. spread into) of the
surrounding local tissues;

 Malignant neoplasms have the capacity to spread
through basement membranes;
 Apart from metastasis, local invasiveness is the most
reliable indicator of neoplasm malignancy

Skin squamous cell carcinoma: carcinoma in situ:
Note that the neoplastic cells have not migrated through the basement
membrane into the dermis i.e. technically, this neoplasm is not invasive

Skin squamous cell carcinoma: local invasiveness:
Note that the neoplastic cells have not migrated through the basement
membrane into the dermis

Definitions
 Metastasis
 Metastasis (i.e. spread to a different anatomical site)
by definition means that a neoplasm is malignant;

 With few exceptions, all malignant tumors can
metastasize;

 In general, the more rapidly a neoplasm grows and
the larger the primary neoplasm, the more likely that
metastasis will occur;

Definitions
 Metastasis
 Pathways of spread include:
 Direct seeding of body cavities or surfaces;
 Lymphatic spread;
 Hematogenous spread;
 Iatrogenic spread

Metastasis to lung of osteosarcoma (tumor of bone)

Definitions

 BY DEFINITION CANCER = MALIGNANT NEOPLASM;

 Carcinogen = A physical or chemical agent that causes
or induces neoplasia;

 Genotoxic carcinogen = carcinogens (or their metabolites)
that directly interact with DNA resulting in mutation;

 Nongenotoxic carcinogen = carcinogens that modify gene
expression but to not directly damage DNA per se.

Definitions

 Key features of genotoxic carcinogens:
 The carcinogen or its metabolites are mutagenic;
 Can act as complete carcinogens or may act as initiators
with subsequent promotion require for neoplasia;
 Neoplasia is dose responsive;
 The neoplastic dose response has no theoretical threshold in
risk assessment terms (biologically, this is very much open to
question and hotly debated)

Definitions

 Key features of non-genotoxic carcinogens:
 Non-mutagenic in classical assays and no direct DNA
damage;
 Neoplasia is dose responsive;
 Neoplastic dose response has a identifiable threshold;
 Effects are reversible if exposure is stopped early enough in
the process;
 May function at the tumor promotion stage;
 Often species, strain, tissue or sex specific.

Multistage Chemical Carcinogenesis:
Classical Concepts

 Core concepts of this hypothesis:

 Carcinogenesis proceeds in a series of definable and
reproducible temporal stages;

 The stages are: initiation, promotion, progression and
outgrowth

7,12-Dimethylbenz[a]anthracene two-hit experimental design in FVB/N mice.

DMBA is a classical initiator; TPA is a classical promoter.
What would you predict the outcome is for each experimental group?

Classical Concepts

 Core features of INITIATION:
 Involves DNA modification/mutation;
 All initiating agents (or their metabolites) are mutagens and
are directly genotoxic;
 DNA damage + one cycle of cell division is necessary to fix
the mutation i.e. cell must survive the DNA damage event
and be able to replicate;
 One single exposure can be sufficient for initiation;
 INITIATION IS IRREVERSIBLE!
 INITATED CELLS APPEAR MORPHOLOGICALLY NORMAL: THE
CHANGES ARE ON THE GENETIC LEVEL AT THIS STAGE!

Classical Concepts

 General features of INITIATORS:
 By definition, either the parent compound or its metabolite
must interact with DNA and produce a definable change in
DNA i.e. a DNA lesion or DNA adduct;
 Most chemical initiators must be metabolized to a DNA
reactive form (i.e. they are indirect initiators or pro-initiators,
or pro-carcinogens). Those that do not (i.e. are direct
acting) tend to be highly reactive chemicals that act at the
site of first contact;
 Many initiators can act as complete carcinogens if the dose
is high enough and repeated exposure occurs. Often what
separates an initiator from a complete carcinogen is DOSE
and REPEATED EXPOSURE.

Classical Concepts

 Initiation is dependent upon MUTATION i.e. the agent must
produce a heritable change in DNA, the cell carrying the
heritable change must survive this change, the cell carrying the
heritable change must avoid necrosis/apoptosis and the cell
carrying the heritable change must be capable of successfully
completing the cell cycle and thus “fixing” the heritable genetic
change;
 The heritable genetic change must not be repaired by the DNA
repair mechanism
 The mutation must be in a genetic location that is favors for
neoplasia while still allowing cell survival and replication;
 The mutation must occur in a stem cell or a cell still capable of
replication rather than cell that has already entered terminal
differentiation;

Classical Concepts

 Once initiation has occurred, there are a number of
possibilities:
 The initiated cell can remain in a static, non-dividing state
through influences by growth control via normal
surrounding cells (paracrine) or endocrine control or
other extrinsic factors;
 The initiated cell lineage may be eliminated because it
has a reproductive &/or survival disadvantage within the
normal tissue;
 The initiate cell may undergo clonal expansion;

Classical Concepts
 Nordling-Knudsen multiple hit theory of carcinogenesis:
 Cancer is the result of accumulated mutations to a cell's
DNA;
 Minimum of 2 “DNA hits” are necessary for carcinogenesis;
 Nordling-Knudsen hypothesis fits well with the more modern
data that demonstrates that for many cancer
types, carcinogenesis (the development of cancer)
depended both on the activation of proto-oncogenes
(genes that stimulate cell proliferation) and on the
deactivation of tumor suppressor genes (genes that keep
proliferation in check);
 However: we now know that there are many more DNA
changes that are needed for malignant neoplasia;
 Indeed, the Nordling-Knudsen hypothesis is probably more
relevant to classical initiation than the entire process of
neoplasia;

Classical Concepts
 In the case of heritable cancers (e.g. retinoblastoma), a
single hit may be all that is required for neoplasia (the first
”hit” being inherited);

 In cancer risk assessment, there is a general assumption
that all genotoxic carcinogenesis operates on the “1 hit”
linear low-dose extrapolation phenomenon that has no
detectable threshold. This is INCREDIBLY CONSERVATIVE;

 Conservatism may be justified in order to protect
vulnerable sub-populations e.g. people who are
heterozygous for tumor suppressor genes (e.g.
heterozygous for mutated p53; mutated TSG’s tend to be
inherited in a recessive manner)

1x10-6
Acceptable Dose=
Slope Factor
DRisk=DDose x SlopeFactor

Classical Concepts

 The counter case against the extreme conservatism of
the “1 hit” carcinogenesis hypothesis in risk assessment
usually follows the following series of hypotheses:
 Promotion and progression of an initiated cell
appear to be relatively rare biological events;
 Promotion is a necessary step for neoplasia;
 Promotion is reversible;
 A substantial number of gene changes are required
for neoplasia, not just changes to protooncogenes
and tumor suppressor genes;

Classical Concepts

 The biological course of neoplasia can be substantially
modified by environmental (i.e. non-genetic) factors;
 There are multiple mechanisms for either repair or removal of
initiated cells;
 There are mechanisms for the safe biotransformation of many
chemical carcinogens;
 Some physical (and chemical??) agents appear to
demonstrate hormesis in their dose response curves (the
classical example being radon daughters)
 All of the above suggest that even in the case of genotoxic
carcinogenesis, there may be a threshold (however for
practical experimental reason, this is very, very difficult to
detect!).

Can you think of some of the implications of the above?
• To detect rare cancer types you need very large numbers of animals;
• At 50 animals per sex per dose, you have a power to detect cancers
incidence of 10%;
• To examine low dose effects, you need very large numbers of animals;
• Conversely, to have adequate ability to detect carcinogenesis we use high
to very high doses, which in turn, forces us to extrapolate beyond actual
experimental measurements.

Classical Concepts
 Attempts at resolving the shape of the low dose cancer
dose response curve – the MEGA studies:
 2 “MEGA” mouse studies;
 1 “MEGA” trout study;

 The results: for the same chemical agent and depending
on the organ and neoplasia type, supralinear, linear and
sublinear dose response curves occur;

 The results: depending on the organ and neoplasia type,
supralinear, linear and sublinear dose response curves
occur in the same animal at the same time!

Classical Concepts
 These results have substantial implications for chemical
carcinogenesis risk assessment:

 In some cases, linear low-dose extrapolation MAY NOT BE AS
CONSERVATIVE AS ORIGINALLY THOUGHT!

 LINEAR LOW-DOSE EXTRAPOLATION MAY SIGNIFICANTLY
OVER OR UNDER-ESTIMATE THE RISK OF CHEMICAL
CARCINOGENESIS AND IN ALMOST ALL CASES, WE HAVE NO
WAY OF KNOWING IF THERE ARE OVER OR UNDER EXSTIMATES
OF RISK;

 THE APPLICATION OF 10-6 ACCEPTABLE RISK TO CANCER +
USE OF THE UPPER CONFIDENCE INTERVAL AS THE POINT OF
DEPARTURE PROVIDES SOME REASSURANCE!

Classical Concepts

 Core features of PROMOTION:
 No direct DNA modification and no direct mutation occurs
i.e. it is a nongenotoxic process;
 Multiple cell divisions leading to clonal expansion of an
individual initiated cell are required;
 Promotion is associated with increased cell proliferation
and/or decreased cell death (i.e. suppression of apoptosis);

Classical Concepts

 Multiple exposures to the promoting agent(s) are required;
 Generally prolonged exposure to the promoting agent(s) is
required;
 Promotion dose response displays a threshold;
 Promotion is REVERSIBLE;

Classical Concepts

 Many (most?) chemical promoters are tissue irritants, but not
all chemical tissue irritants are promoters!
 Many (most?) promoters stimulate cell proliferation (can be
multiple different mechanisms ranging from simple
damage/repair responses to receptor-mediated
endocrine/paracrine effects;
 INITIATION MUST PRECEED PROMOTION! PROMOTION BY
ITSELF (IN THE ABSENCE OF INITATION OR PRIOR TO
INITIATION) IS NOT SUFFICIENT FOR CARCINOGENESIS!
 The outcome of promotion is a pre-neoplastic lesion.

Classical Concepts

 Features of promotion and chemical promoters:
 Chemical tumor promoters are not mutagenic i.e. they do
not produce direct DNA lesions or DNA adducts;
 Chemical tumor promoters act via mechanisms that either
modify gene expression and/or stimulate sustained cell
proliferation;
 EFFECTS OF PROMOTERS ON CELL PROLIFERATION ARE TIME
LIMITED I.E. REPEATED AND (OFTEN) PROLONGED EXPOSURE
TO THE PROMOTING AGENT IS REQUIRED FOR
CARCINOGENESIS;

Classical Concepts

 IF PROMOTION IS STOPPED, CLONAL EXPANSION STOPS AND
THE EXPANDED CELL POPULATION IS ELIMINATED VIA
APOPTOSIS I.E. PROMOTION IS REVERSIBLE;
 PROMOTION IS A THRESHOLD EFFECT I.E. DOSE RESPONSE
THRESHOLDS CAN BE DETECTED! This is because there are
doses below which cell proliferation is not stimulated.

Classical Concepts

 Tumor promoters can be endogenous or exogenous;
 Many chemical tumor promoters have an equivalent
endogenous ligand;
 Many chemical/endogenous tumor promoters are organ,
tissue or cell type-specific e.g. phenobarbital is a tumor
promoter in rodent liver, but not in the skin;
 Many non-genotoxic carcinogens appear to act as tumor
promoters or at the tumor promotion stage of
carcinogenesis.

Classical Concepts

 Key features of progression:
 Characterized by increasing genetic instability
characterized by continued mutation, and chromosomal
disarrangement;
 No further exogenous or endogenous stimulus is required for
continued expansion of transformed cells i.e. the cells have
become imortalized, self-sufficient and resistant to signals
that are inhibitory for growth.
 However, the neoplastic cell population can still be
influence by tissue/endogenous/exogenous stimuli;

Classical Concepts

 Immunoescape is important: avoidance of destruction by
the innate and adaptive immune system;
 The neoplastic cell population is morphologically
identifiable: “carcinoma in situ”;
 A key feature of progression is that neoplasms become
progressively more anaplastic, progressively more anaplastic
and their cell population becomes progressively more
heterogenous;

Classical Concepts

 Progression is associated with the accumulation of multiple
mutations that accumulate independently in different cells.
 The independent accumulation of new mutations in
different cells results in the formation of sub-clones of cells;
 Even though almost all neoplasms are monoclonal in origin,
by the time that they are clinically detectable, their
constituent cells are extremely heterogenous;

Classical Concepts


 ONCE PROGRESSION HAS STARTED IT IS IRREVERSIBLE;

 CHEMICALS THAT ARE PROGRESSOR AGENTS TEND TO BE
GENOTOXIC AND ARE TYPICALLY CLASTENOGENIC;

Classical Concepts

 Key features of outgrowth:
 Invasion: the ability of neoplastic cells to migrate through
basement membranes or other tissue barriers
 Angiogenesis: in order to expand beyond a diameter of 1 –
2 mm, the neoplastic cell population must obtain a blood
supply by the stimulating new blood vessels
(neovascularization);
 Immunoescape remains important;
 Formation of a favorable extracellular matrix or favorable
modification of the extracellular matrix;
 Metastasis.

Molecular Basis of Carcinogesis:
Fundamental Principles

 Non-lethal genetic damage (mutation) is the foundation
of carcinogenesis;
 Source of mutation can be environmental (chemical,
physical or infectious [viruses]);
 Source of the mutation can be inherited;
 Can be due to chance (spontaneous and stochastic);

 A neoplasm is formed by the clonal expansion of a single
precursor cell that has incurred genetic damage (i.e.
neoplasms are monoclonal);


 5 basic classes of normal cell regulatory genes are the
principal targets for procarcinogenic genetic damage:

 Growth-promoting proto-oncogenes;
 Growth-inhibiting tumor suppressor genes;
 Genes that regulate apoptosis/programmed cell death;
 Genes that regulate terminal differentiation;
 Genes involved in DNA repair


 Proto-oncogenes and oncogenes:
 A mutant allele of a proto-oncogene is called an
oncogene;
 Oncogenes tend to inherited in a dominant pattern i.e. cells
which are heterozygous display the dominant oncogene
phenotype;


 Proto-oncogenes and oncogenes:
 Since oncogenes are dominant, it follows that only a single
mutation is required to convert a proto-oncogene
phenotype to an oncogene phenotype;

 Loss of gene function due to a single mutated allele is called
haploinsufficiency;


 Tumor suppressor genes:
 Typically, both alleles of a tumor suppressor gene must be
mutated before a loss of function (i.e. a pro-neoplasia
phenotype) occurs;
 If follows that (a) mutated tumor suppressor alleles are mostly
inherited in a recessive manner; and (b) a minimum of two
mutagentic events (i.e. mutation of both tumor suppressor
gene alleles) is required before a pro-neoplasia phenotype
occurs;1
1: Not entirely true in all cases as genetic variable penetrance is also
present in some cases i.e. the heterozygous phenotype is intermediate
between that of the 2 homozygous phenotypes; or the loss of 1 functional
allele may be sufficient to produce a pro-neoplastic phenotype.


 Genes regulating apoptosis/programmed cell death
may behave like proto-oncogenes or tumor suppressor
genes in terms of their genetic:phenotype relationships;


 Mutations in DNA repair genes affect cell proliferation
indirectly by modifying the ability of the cell to repair
dangerous mutations in proto-oncogenes, tumor
suppressor genes and the genes that regulate apoptosis;
 Cells with defective DNA repair function have a “mutator
phenotype”;


 Carcinogeneis is a multistep process both at the
phenotypic and genetic levels;

 Carcinogenesis requires accumulation of multiple
mutations in specific locations;
 A single mutation is rarely (never?) sufficient for
carcinogenesis;
 Pro-carcinogenic mutations are not random throughout the
genome: the mutations must be in the right places in the
right genes;

Essential Genetic/Phenotypic
Alterations for Malignancy

 The following are the fundamental requirements for
neoplasia and malignancy (progression and outgrowth):
 Self-sufficiency in growth signals i.e. no exogenous signals or
stimuli are required for continued cell division;
 Insensitivity to growth-inhibitory signals e.g. not responsive to
TGFβ or inhibitors of cyclin-dependent kinases;
 Evasion of apoptosis: e.g. by the inactivation of p53 or
activation of anti-apoptosis genes;


 Attained limitless replicative potential (i.e. become
imortalized) i.e. avoid senescence, terminal differentiation
and mitotic catastrophe;
 Immunoescape: avoidance of the host innate and adaptive
immune systems;
 Induction of sustained angiogenesis;


 Gained favorable interaction with the extracellular matrix;
 Gained the ability to invade and metastasize;
 Gained defects in DNA repair

Self-Sufficiency in Growth Signals:
Proto-Oncogenes, Oncogenes, Oncoproteins

 Fundamental concept: the mutation of a proto-oncogene
to an oncogene and the production of the altered
oncoprotein promotes cell growth and replication in the
absence of growth factors and/or other external stimuli i.e.
cell growth becomes autonomous and oncogenes allow
cells to be self-sufficient in terms of growth signals;

 Proto-oncogene proteins can function as:
 Growth factors;
 Growth factor receptors;
 Signal transducers;
 Transcription factors;
 Cell cycle components.

Generalized normal steps in signaling of
cell growth and replication (using a
simplified RAS schematic as an
example):

• Binding of a growth factor to its cell
membrane receptor;

• Transient and limited activation of the
relevant growth receptor and signal
transduction on the inner leaflet of
the plasma membrane;

• Transmission of the transduced signal
across the cytoplasm;

• Induction and activation of nuclear
regulatory factors that initiate DNA
transcriptoion;

• Entry, progression and completion of
the cell cycle  cell division

Oncogenes can act at any of these levels

Types of Oncogenes

 Growth factor genes;

 Growth factor receptors;

 Genes for proteins involved in signal transduction;

 Genes for nuclear regulatory proteins;

 Genes for cell cycle regulators.

Types of Oncogenes: Growth factor genes

 The most common modes of action of growth factor
oncogenes is overexpression or amplification;

 Most growth factors normally act in a paracrine manner
(i.e. produced by one cell type, and act locally on a
neighboring cell);

 Many transformed cells acquire the ability to synthesize
growth factors to which they then respond


 In most instances, the growth factor genes are not
altered;

 The most common mode of action is that the signal
transduction pathway that activates synthesis of the
growth factor is corrupted (i.e. permanently switched on)
i.e. the gene is over expressed;

 Permanent growth factor production creates a pro-
malignant phenotype because increasing the number of
cell divisions increases the risk of further stochastic (i.e.
chance) mutations


 Important examples:
 TGFα; (TGF = tissue growth factor)
 PDGF-βchain; (PDGF = platelet derived growth factor)
 Fibroblast-derived growth factors (HST1, FGF3);
 HGF; (hepatocyte growth factor)

Types of Oncogenes: Growth factor receptors

Many of the critical GFR
exist in the non-activated
state as monomers, with
the inactive tyrosine
kinase enzyme being
bound to the inner leaflet
of the cell membrane

Activation occurs by 2
signal molecules binding
to two nearby Tyrosine-
Kinase Receptors, causing
them to aggregate,
forming a TRANSIENT
dimer. This activates the
tyrosine kinase, which in
turn, uses ATP to
phosphorylate (i.e.
activate) a second
messenger protein, which
in turn, triggers the
downstream signal
transduction/messaging
cascade.


 Oncogenic forms of the GFR are associated with
constitutive, PERMANENT, dimerization in the absence of
the receptor ligand i.e. the receptor is permanently
switched on irrespective of whether or not the normal
growth factor is present;

 The permanently activated tyrosine kinase is the target of
the anticancer drug, imatinib mesylate;

 Permanent switching on of growth factor receptors can
occur by simple point mutations, translocations or over-
expression mechanisms.


 Important example: the RET proto-oncogene
 Encodes a receptor tyrosine kinase for members of the glial
cell line-derived neurotrophic factor family of extracellular
signalling molecules;
 Gain of function by mutation results in thyroid and other
endocrine carcinomas;
 2 important point mutations: MEN-2A and MEN-2B


 Important example: the RET proto-oncogene
 The MEN-2A point mutation cause constitutive dimerization
of the receptor (i.e. the receptor is permanently and
erroneously switched on)  switching on of the TGF-
βpathway;
 The MEN-2B point mutation alters the substrate specificity of
the tyrosine kinase which results in exaggerated TGF-
βsignalling;
 Both the MEN-2A and 2B oncogenes are inherited as
autosomal dominants i.e. only one allele needs to be
altered for the proneoplastic phenotype.

Types of Oncogenes: Signal transduction proteins

 THE classical example is the RAS proto-oncogene family
(HRAS, KRAS, NRAS) !;

 Point mutations in members of the RAS family are the
most common mutations in human cancers: about 15-
20% of all human cancers carry them;

 As a general rule:
 Cancers of the bladder have HRAS mutations;
 Carcinomas have KRAS mutations;
 Hemopoeitic neoplasias have NRAS mutations.

RAS "molecular switch": GTP-bound form is active while the GDP-bound
form is inactive. In its active form, RAS interacts with its effectors. This
cycling is regulated by GTPase-activating proteins (GAPs) and
nucleotide exchange factors (GEFs).

One of the most studied RAS-controlled pathways is the RAS/MAPK pathway. This is one of
the most conserved pathways throughout evolution, and controls biological processes
such as proliferation, differentiation, apoptosis, and migration. The directionality of
signaling from extracellular signals to effector proteins is regulated by the on/off switch
states of RAS proteins. Any mutation of RAS affects the on/off balance and RAS mutations
at codon 12, 13 and 61 lead to constitutive active RAS protein and the subsequent hyper-
activation of the MAPK pathway.


 Mutations that result in permanent switching on of RAS
involve:
 Changes in the GTP-binding pocket or the enzymatic region
responsible for GTP hydrolysis  net result is to decrease the
GTPase activity of RAS  keeps RAS in the GTP-RAS
activated form;

 Mutation of the GAP proteints  fail to activate the RAS-
GTPase activity  keeps RAS in the GTP-RAS activated form;

 Mutation and permanent activation of down stream signal
components in the RAS/RAF/MAP kinase pathway
(important in melanoma);


 Oncogene-induced senescence:

 in wild-type cells, activated RAS triggers an initial wave of
proliferation, followed by an irreversible growth arrest known as cellular
senescence and a concomitant accumulation of p53 and p16 proteins
 apoptosis;

 In melanoma, the presence of BRAF oncogenes in the absence of
dysregulation of p53/p16 results in benign melanocytic nevi rather than
melanoma;

 Demonstrates that the presence of an oncogene by itself is not sufficient
for neoplasia. Other gene mutations (notably in the tumor suppressor
genes p53 and/or p16) are required for carcinogenesis;

Types of Oncogenes:
Non-receptor tyrosine kinases

 NRTKs normally function in the signal transduction
pathways that regulate cell growth;

 ABL kinases
 c-ABL is associated with leukemias;

Growth factors (GF) induce SFK activation in caveolae, allowing
phosphorylation of c-Abl to increase its catalytic activity. c-Abl then
activates Rac/JNK and Rac/NADPH oxidase (Nox) pathways to induce c-
myc expression, a transcription factor required for induction of DNA
synthesis.. This set of events promotes G1-phase progression of the cell
cycle, leading to S-phase entry and DNA synthesis..

Types of Oncogenes:

 ABL chromosomal translocation results in movement of
the ABL gene from chromosome 9 to chromosome 22
forming a ABL-BCR hybrid gene;

 The ABL-BCR hybrid oncogene produces a ABL-BCR
hybrid fusion protein that has increased kinase activity 
chronic myelogenous leukemias;

 Formation of similar tyrosine kinase fusion proteins and
hybrid tyrosine kinase oncogenes are a common pattern
in a number of forms of neoplasia

Types of Oncogenes:

 Imatinib mesylate
 Anti-cancer drug used to treat CML;
 Specifically targets BCR-ABL kinase activity
 Works on the basis that in many forms of CML, continuous
signaling down the BCR-ABL kinase pathway is necessary for
persistence of the transformed cells and the neoplasia.
Disrupting this signaling results in death of the neoplastic
cells;
 Actions of this drug demonstrate that the BCR-ABL kinase
oncogene is a lynch-pin mechanism in CML and possibly
other types of leukemias.

Types of Oncogenes: Transcription factors.

 A large number of oncogenes fall into this category
(MYC, JUN, FOS, MYB, REL);

 The MYC oncogene is the one most commonly involved
in human neoplasias;

 MYC is present in virtually every eukaryotic cell type;

 MYC regulates a huge number of genes and cell
functions;

 Belongs to the “early response genes” that are induced
when resting cells receive a signal to divide;

Types of Oncogenes: Transcription factors.

 MYC is normally transiently switched on via a variety of
cell signals (predominantly those which trigger cell
division);

 Mechanisms of MYC overexpression:
 Coupled with a more active promoter by chromosome
translocation (Burkitt’s lymphoma)
 Removal of the 3’ UTR destabilizing sequences, resulting in
an elevation of MYC mRNA;
 Insertion of retroviruses adjacent to the MYC locus activates
its expression via retroviral regulatory sequences
 Oncogenic RAS appears to stabilize the MYC

Types of Oncogenes:
Cyclins and cyclin-dependent kinases.

 The normal and orderly progression of cells through the
cell cycle (i.e. replication) is orchestrated by cyclin-
dependet kinases (CDKs);

 CDKs are activated by binding to cyclin proteins
 Cyclins are “cyclic” in the sense of their production and
degradation;

Types of Oncogenes:
Cyclins and cyclin-dependent kinases.

 Bottom lines:
 Mutations that dysregulate the activity of cyclings and CDKs
favors cell proliferation;
 Common targets in neoplasia are cyclin D and CDK4 (i.e.
targeting the G1 phase of the cell cycle);
 Cyclin D is overexpressed in many neoplasias;
 CDK inhibitor (e.g. p16, p21, p27, p57) expression is
commonly reduced or absent  favorable for cell
proliferation e.g. germline mutations of p16 are present in
a human sub-population that is prone to melanoma;

Cell Cycle Checkpoints

 Defects in the cell cycle checkpoint system are a major
cause of genomic instability in neoplastic cells!

 G1/S transition check point
 Checks for DNA damage;
 If DNA damage is present, the DNA repair mechanisms are
switched on;
 Provides time for DNA repair to occur;
 Allows entry into apoptosis if the cellular damage is too
extensive.


 G2/M checkpoint
 Monitors the completion of DNA replication
 Checks whether or not the cell can safely initiate mitosis and
separation of sister chromatids
 Particularly important in ionizing radiation damaged cells
and if chromosomal damage is present.


 There are a series of cellular sensors and transducers for
the cell cycle checkpoints;

 G1/S checkpoint is mostly mediated by p53 which acts
via the cell cycle inhibitor p21;

 G2/M checkpoint is mediate by p53 dependent and p53
independent mechanisms.

Escape From Growth Inhibition and Senescence:
Tumor suppressor genes

 Fundamental concept: oncogenes drive the proliferation
of cells where as tumor suppressors apply the brakes to
proliferation OR their activation triggers post-mitotic cell
differentiation (i.e. entry into the differentiated cell pool
without replicative potential);

 Many TSGs are part of a network of sensors that detect
cellular genotoxic stress from just about any source;

 Function is to shut down cell proliferation;

 Remember the ocogene senesce paradigm:

Oncogene-Induced Senescence

 Oncogene-induced senescence:

 in wild-type cells, activated RAS triggers an initial wave of proliferation,
followed by an irreversible growth arrest known as cellular senescence
and a concomitant accumulation of p53 and p16 proteins  apoptosis;

 In melanoma, the presence of BRAF oncogenes in the absence of
dysregulation of p53/p16 results in benign melanocytic nevi rather than
melanoma;

 Demonstrates that the presence of an oncogene by itself is not sufficient
for neoplasia. Other gene mutations (notably in the tumor suppressor
genes p53 and/or p16) are required for carcinogenesis;

Escape From Growth Inhibition and Senescence:
Tumor suppressor genes

 TSGs are generally autosomal recessive i.e. for the
defective phenotype to occur, the cell must carry both
mutated alleles;

 In general, 2 mutation events are required for the most
defective phenotype to occur;
 At least one, or both, of the mutant alleles can be inherited
 If the cell is already heterozygous, only 1 additional mutation
is required;

 Limited penetrance may operate in many cases i.e. the
hetrozygos cell or individual may have a phenotype that
lies between the two homozygous phenotypes.

Tumor Suppressor Genes:
Retinoblastoma protein

 RB regulates the G1/S checkpoint of the cell cycle

 RB is one of the classical tumor suppressor genes;

 Inherited as an autosomal recessive allele with a
mutation at locs 13q14;

 The dominant normal RB allele has full penetrance i.e.
heterozygous cells are otherwise completely normal;


 Familial Retinoblastoma:
 Once defective allele is typically inherited;
 The second defective allele is typically acquired by
mutation;

 Random retinoblastoma:
 2 mutations are required because 2 normal RB alleles are
present (one from each parent);

Normal functioning of the retinoblastoma protein (RB)

1. In the G0 phase, S-phase-
Normal RB Protein Function: specific genes are not
transcribed because of the
inhibitory effect of the RBR
protein on the E2F/DP
heterodimeric complex.

2. When cells are committed
to divide, D-type cyclins
(CYCD) will be transcribed
in G1 and will start to form
active complexes with G1–
S-specific CDK proteins
(CDKA). The active CDK–
cyclin complex
phosphorylates the RBR
protein, releasing the
E2F/DP complex.

3. Subsequently, the active
E2F/DP complex will
activate transcription of
genes necessary for DNA
replication.


 If the RB protein is lost or is defective:
 The G1/S checkpoint no longer functions;
 There are no longer adequate checks for DNA
damage;
 The DNA repair mechanisms are either not switched
on or are faulty;
 There is insufficient time for DNA repair;
 The overall net result is increased genetic instability
and loss of the ability of the cell to detect and repair
subsequent mutations or to eliminate transformed cells
via apoptosis.


 Critical sites for RB protein mutation:
 Mutations are localized to an area of the protein
called the RB pocket;
 The RB pocket is involved in the binding of RB to E2F;


 RB protein/gene may be normal but loss of RB function
can still occur:
 Mutations in the proteins that phosphorylate RB will
mimic RB loss e.g. mutational activation of CYCA and
CYCD results in permanent RB phosphorylation 
transcription is permanently switched on
 Many cancers that do not have RB gene abnormalities
will have a functional loss of RB inhibition excessive
phosphorylation of RB due to CDK mutations.


 Current critical paradigm:
 At least one of the 4 major regulators of the cell cycle
(p16/INK4a, RB, Cyclin D, CYKA) are dysregulated in
some form in almost all human cancers,

Tumor Suppressor Genes: p53

 p53 is the guardian of the genome;

 P53 acts at the G1/S checkpoint of the cell cycle;

 Inactive or dysregulated p53 is amongst the most
common deficits in human cancer;

 50% of human cancers contain p53 mutations;


 Homozygous loss of p53 function occurs in almost all
human cancers;

 In most cases, loss of p53 function is AQUIRED;

 Rarely, one defective p53 allele may be inherited (Li-
Fraumeni Syndrome);


 Normal p53 regulation:
 In non-stressed cells, p53 has a short half-life (~ 20
minutes);
 The short half-life is due to the association of p53 with
MDM2;
 MDM2 inhibits p53 activity because it blocks its
transcriptional activity, favors its nuclear export and
stimulates its degradation


 Normal p53 regulation:
 miRNAs (mir34a-mir34c)
 miRNAs bind to mRNA preventing its translation i.e.
block protein synthesis
 mir34 thus mimic many of the functions of p53


 p53 acts to prevent neoplasia by 3 critical mechanisms:
 Activation of temporary cell cycle arrest (quiescence; allows
for repair);
 Induction of permanent cell cycle arrest (senescence);
 Triggering of apoptosis;

 p53 activation is the primordial mechanism for initiating
DNA repair;


 How does p53 sense cellular stress and DNA damage?
 At least 2 proteins appear to act as biosensors for DNA
damage: ataxia-telangiecasia mutated protein kinase
(ATM) & ataxia-telangiecasia and RAD3 related protein
kinase (ATK);
 Once damage is sensed, ATR and ATK phosphorylate p53,
which activates it.

 Raises the specter that functional p53 loss could occur if ATM
and ATK are rendered dysfunctional by mutation;

the APC/β-catenin pathway

 APC is a class of tumor supresssor genes which down-
regulate growth promoting signals;

 APC is a typical TSG i.e. 2 mutant alleles are required for
the pro-neoplastic phenotype;

 Germ-line mutations in APC are associated with familial
adenomatous polyposis (pre-disposes to colon cancer);


 70-80% of human colorectal cancers display the
homozygous loss of APC function;

 Forms part of the WNT signaling system;

When the WNT receptor is not occupied,
Axin, GSK and APC form a "destruction
complex," and β-Cat is destroyed.

• When the WNT receptor is occupied, Axin is
removed from the "destruction complex." β-Cat
moves into the nucleus, binds to a transcription
factor on DNA, and activates transcription of a
protein. "P" represents phosphate;

• With the pro-neoplastic loss of APC function, APC
can no longer mediate the cytoplasmic
destruction ofβ-catenin  the WNT pathway is
permanently switched on;

• The alternative mechanism which also results in
permanent activation of the WNT pathway are
mutations in β-catenin which render it resistant to
destruction by APC.


 An additionally critical effect of the cellular
accumulation of β-catenin is that this protein is also
necessary for normal cell to cell adhesion;

 β-catenin is also normally bound to the cytoplasmic tail
of E-cadherin which is responsible for intercellular
adhesiveness;


 When cells are torn apart (injury), β-catenin is released
from the membrane bound E-cadherin into the
cytoplasm  nucleus  transcription;

 When cell to cell contact is re-established , β-catenin is
again sequestered by binding to cell membrane E-
cadherin and nuclear transcription ceases. This is the
fundamental mechanism of contact inhibition of normal
cells;


 Disruption of the E-cadherin-β-catenin pathway is a
critical event in neoplasia and leads to the classical loss
of contact inhibition seen in the malignant phenotype;

 Disruption of the E-cadherin-β-catenin pathway is a
critical event that allows for migration, invasiveness and
metastasis of malignant neoplastic cells.

the TGFβ pathway.

 In epithelial and hematopoeitic cells, TGFβis a potent
inhibitor of cell proliferation;

 TGFβsignaling is also an important pathway for the
induction of apoptosis;
 There are 2 known mechanisms: the SMAD pathway & the
DAXX pathway;

 In many forms of cancer, the growth-inhibiting effects of
the TGFβ pathway are lost by mutations in the signaling
pathway (100% of pancreatic cancers, 83% of colon
cancers).

Evasion of Apoptosis.

 Activation occurs by 2 different pathways:
 Extrinsic pathways acting via death receptors. These
receptors activate Death Caspases within seconds of
ligand binding, causing an apoptotic demise of the cell
within hours.
 Via CD95/Fas signaling;
 Via CD120/tumor necrosis factor receptor 1 signaling;
 Apo 2 and Apo 3

 Activation occurs by 2 different pathways:

 Intrinsic pathway following DNA and/or mitochondrial damage;
 The core event is alteration of mitochondrial permeability
and loss of mitochondrial transmembrane potential (which
results in loss of funciton of the electron transport chain and
loss of ATP production);
 Altererd mitochondrial permeability results in CytoC release
and formation of the Apoptosome, a catalytic multiprotein
platform that activates Caspase9. Activated Caspase9 then
cleaves Caspase3 resulting in downstream events involved
in cell death;
 Release of CytoC is regulated by Bcl2 family proteins which
reside in the outer mitochondrial membrane and prevent
CytoC release.

 The ability to evade apoptosis following mutation is a key event in
carcinogenesis;

 Known mechanisms of evasion include:
 Loss or downregulation of FAS/CD95;
 Induction of a protein called FLIP which prevents the activation
of caspase 8;
 Over-expression of BCL-2;

 Over-expression of Bcl-2:
 Best described mechanism;
 Bcl-2 derives its name from B-cell lymphoma 2;
 Mechanism of over expression involves translocation of the Bcl-
2 gene on chromosome 18 to the immunoglobulin promoter
region of chromosome 14;
 Net result is that a normally tightly regulated gene is moved to
a area of chromosome 14 that is more actively transcribed,
with the result that Bcl-2 is over-produced;

 Over-expression of Bcl-2:
 Present in ~85% of B-cell lymphomas;

 Bcl-2-induced lymphomas largely result from a failure of
apoptosis rather than an explosive proliferation. This explains
their behavior – these cancers tend to be slow growing
compared with other types of lymphomas.

Evasion of the Immune Response.
 A large array of strategies are used:
 Tumor cells are often not antigenic i.e. not recognized by the
immune system. This is driven by active immunoselection in
most cases;
 In some cases, the carcinogenic process is immunosuppressive
or induces immune-tolerance to the transformed cells (e.g. UV
radiation-induced non-melanoma skin cancer);
 Loss of MHC molecules and other antigen presenting molecules
from the cell surface  cells cannot be detected by cytotoxic
T-cells;

 Tumors often secrete proteins that inhibit effector T cell
responses and promote the production of regulatory T cells that
suppress immune responses;

 Certain melanomas can reorganize their stromal
microenvironment (the supportive connective tissue) into
structures similar to lymphoid tissue of the immune system. This
ingenious reconstruction recruits and maintains immune
regulatory cells that promote tolerance and tumor progression;

 Tumor cells can produce receptor types (e.g. Toll receptors)
that facilitate immune evasion.

 Neoplastic cell TGFβsecretion inhibits NK cell function;

 Neoplastic cells can become resistant to immune-mediated
induction of apoptosis;

 Many tumors, particularly when the breach the body
surface, contain areas of active infection;

 This is a very active area of study: there are many other
proposed mechanisms

Limitless Replicative Potential: Telomeres and
escape from mitotic catastrophe
 Normal human cells have the capacity for 60 to 70 replications
(the Hayflick limit) after which, they loose their ability to divide 
senescence;

 The loss of the ability to replicate is because of progressive
shortening of the telomeres present at the ends of chromosomes;

 Short telomeres  behave like DNA double strand breaks 
triggers p53 and RB-mediated cell cycle arrest at the G1/S
checkpoint;

 When the G1/S checkpoint is disabled (i.e. p53 and RB are
dysfunctional):
 When the cell replication is still possible via chromosomal non-
homologous end joining;

 Non-homologous end joining results in dicentric chromosomes;

 When dicentric chromosomes are pulled apart during the
anaphase of mitosis, new double stranded DNA breaks are
created  the net result is substantial genomic instability 
multiple bridge-fusion cycles results in a mitotic catastrophe 
cell death

 For a neoplastic cell to survive and thrive it must:
 Evade the shortening of telomeres AND
 Avoid the mitotic catastrophe;

 Successful neoplastic cells accomplish this by reactivating
telomerases (normally only active in embryonic stem cells).

 There are other alternative mechanisms for lengthening telomeres
which are calledalternative lengthening of telomeres (ALT);

 The induction of telomerases in neoplastic cells gives them endless
replicative potential i.e. they become immortal.

Getting adequate blood supply: Angiogenesis

 Neoplasias cannot grow beyond a diameter of ~1-2 mm without
inducing a new blood supply;

 Neoplastic cells stimulate the creation of new blood vessel
branches from previously existing blood vessels. This involves
recruitment of normal endothelial cells from the bone marrow;

 The vasculature in neoplasias is abnormal: leaky, inefficient,
haphazard branching;

Getting adequate blood supply: Angiogenesis

 Neovascularization has 3 major effects:
 Provides nutrients required for continued growth;
 Newly formed endothelial cells secrete growth factors (notably
insulin-like growth factor, platelet-derived growth factor, GM-
CSF) which favor neoplastic cell growth;
 Provides access to the vascular system which is necessary for
metastasis;

 Neovascularization is a necessary biological correlate for
malignancy.

Growth factors involved in cancer angiogenesis/neovascularization

Spreading The Love: Invasion and Metastasis

 Neoplastic cells by their very nature often have poor cell to cell
adhesion  millions of neoplastic cells are regularly released into
the circulation during carcinogenesis, however metastases are
frequently relatively uncommon;

 The metastatic cascade consists of 2 basic phases:
 Invasion of the extracellular matrix;
 Vascular dissemination, homing and colonization.


 Invasion of the ECM consists of 4 major steps:
 Reduction or loss of cell to cell adhesion;
 Degradation of the ECM;
 Attachment to novel ECM components;
 Migration of neoplastic cells


 Cell to cell dissociation:
 Disruption of the catenin-E-cadherin system (discussed
previously);


 Degradation of the basement membrane and interstitial
connective tissue:
 Neoplastic cells secrete proteolytic enzymes (many different classes
including matrix metalloproteases, cathepsin D, urokinase
plasminogen activator);
 Neoplastic cells induce connective tissue damage by stimulating
normal stromal cells (fibroblasts, myofibroblasts);


 Degradation of the basement membrane and interstitial
connective tissue:
 MMP activity also releases growth factors which stimulate neoplastic
cell growth;
 Loss of polarity and contact inhibition in neoplastic cells means that
they are no longer dependent on attachment to the ECM for survival
and growth.


 Locomotion and migration:
 Invasion involves the movement of neoplastic cells through
damaged basement membranes or through zones of disrupted
ECM
 Locomotion of cells is an extremely complex process involving
many different types of receptor and cellular systems;
 Locomotion/migration in neoplastic cells is potentiated and
driven by autocrine production of motility factors plus damage
to the ECM (e.g. cleavage of collagen and laminin).


 Vascular dissemination and homing
 Single cells in the circulation are prone to damage by
mechanical and fluid-dynamic mechanisms i.e. the circulation
is an environment that is not favorable to the survival of single
neoplastic cells;

 Single cells in the circulation are prone to destruction by the
immune system;

 Within the circulation neoplastic cells tend to form cell
aggregates, cell-platelet or activate the clotting cascade to
form true emboli, all of which provide protection;


 Vascular dissemination:
 The sites that neoplastic cell aggregates/emboli eventually
lodge is often dependent on the site of the primary tumor i.e.
metastases commonly form in the first capillary bed that the
tumor cells encounter after entry into the circulation (lung, liver,
brain etc);

 Natural vascular drainage patterns do not explain all the types
of metastatic distribution encountered – specific homing to
particular tissues appears to operate in some cases;


 Homing
 Selective tissue homing can be explained in some cases by the
presence of particular cell surface adhesion molecules and
target ligands in the vascular beds of the target tissue;

 Chemokines an chemoattractants play a critical role in other
cases.


 Paget’s “fertile soil” or “seed and soil” hypothesis:
 Proposed in 1889;
 The hypothesis is considered a milestone in cancer biology and
pathology;
 Core concepts:
 The sites of secondary growths are not a matter of pure chance;
 The sites of secondary growths are not just a matter of the degree
of vascularization and perfusion e.g. skeletal muscle is vey vascular
and well-perfused, yet metastasis in skeletal muscle is very rare;
 Many neoplastic cells that lodge in distant tissues either die
(apoptosis) or they remain dormant and do not develop;
 Some organs provide a more fertile environment than others for
the growth of certain metastases.


 Paget’s “fertile soil” or “seed and soil” hypothesis:
 A constant pattern is that the neoplastic cells themselves
modify the resident stromal cells of the receiving tissue to
create a more habitable site for metastasis.

Genomic Instability: the enabler of malignancy

 Humans literally swim in a myriad of agents that are mutagenic,
however cancers are relatively rare outcomes;

 The reason for this is the ability to repair DNA or kill off cells with un-
repairable DNA damage combined with the ability of the innate
(and sometimes the adaptive) immune system to selectively
target and destroy transformed cells;


 Individuals born with deficits in DNA repair are extremely prone to
neoplasia:

 In a sense, most DNA repair genes operate like TSG – i.e. both
alleles need to be inactivated before there is an increase in
neoplasia (although limited penetrance and intermediate
phenotypes are important in some cases);


 Hereditary Nonpolyposis Colon Cancer Syndrome
 Due to a inherited loss of DNA mismatch repair;
 Hallmark is microsatellite instability
 Microsatellites are also known as Simple Sequence Repeats
(SSRs) or short tandem repeats (STRs), are repeating
sequences of 2-6 base pairs of DNA;
 In normal individuals, their location and base pair
composition is usually very stable;

 Individuals with this genotype have very high rates of colon
cancer.


 Xeroderma pigmentosum
 Autosomal recessive genetic disorder of DNA repair in which the
ability to repair damage caused by ultraviolet (UV) light is deficient;
 Primary defect is in DNA excision repair;
 Cannot repair T-T dimers and 6-4 photoproducts in skin;
 Extremely prone to non-melanoma skin cancer


 Defects in DNA repair due to chromosome homologous
recombination:
 Blood syndrome, ataxia-telangiecasia, Fanconi anemia

The Warburg Effect

 Even in the presence of ample oxygen, neoplastic cells are
heavily dependent upon anerobic glycolysis rather than
mitochondria for energy production;

 Cancers have very high glucose requirements compared with
normal tissues (taken advantage of in some imaging and drug
targeting regimes [PET imaging]);

 Warburg effect is assumed to provide neoplastic cells a growth
advantage in relatively hypoxic tumor environments (despite
angiogenesis, most cancers remain relatively hypoxic);

The Warburg Effect

 The effect is mediated by the loss of functional p53:

• In normal cells,
glucose is actively
transported into cells;
• Under hypoxic
conditions, hypoxia-
inducible factor 1
(HIF1) and MYC
collaborate to
activate hexokinase
2 (HK2) and pyruvate
dehydrogenase
kinase 1 (PDK1),
resulting in
enhanced
conversion of
glucose to lactic
acid;
• HIF1 and MYC
independently
activate the glucose
transporter GLUT1
and lactate
dehydrogenase A
(LDHA).

Nongentoxic (Epigenetic)
Carcinogens

Nongenotoxic Carcinogens:
Fundamental concepts.

 Nongenotoxic carcinogen = agents that induce neoplasia without
direct DNA binding, damage or interaction of either the primary
agent or its metabolites;

 Organ and tissue targets of nongenotoxic agents tend to be those
where there is normally a significant background level of
carcinogenesis in non-exposed animals (e.g. liver in mice);

 Generally speaking, prolonged exposure to high doses of
nongenotoxic agents is necessary for neoplasia;

Fundamental concepts.

 Nongenotoxic agents are assumed to have a dose threshold
below which neoplasia will not occur;

 Nongenotoxic carcinogenesis is often species dependent and
can be sex dependent.

Key mechanisms.

 Fundamental concept: any mechanism that results in sustained
and excessive cell proliferation is potentially associated with
nongenotoxic carcinogenesis;

Key mechanisms.

 Known mechanisms are:
 Cytotoxicity + regenerative hyperplasia;
 α2u-Globulin binding in male rats;
 CAR receptor mediated;
 Peroxisome proliferation;
 AhR mediated
 Hormonal (biogenic amines, steroid and peptide hormones, DES,
phytoesrogens, tomoxifen, pheobarbital)
 Altered methylation (epigenetic change; diethanoloamine, choline
deficiency, phenobarbital);
 Inducers of oxidative stress (ethanol, TCDD, lindane, dieldrin,
acrylonitrile).

Cytotoxicity + regenerative hyperplasia
(cytolethality).

 Best known examples:
 Chlorinated hydrocarbon-induced renal tumors in rodents (e.g.
chloroform);
 Chloroform
 Melamine-induced bladder tumors in rodents;

 Mechanisms:
 High rates of cell replication increase the risk of spontaneous
mutations;
 Increased speed of replication reduces the odds of cell cycle arrest
and the G1/S checkpoint;
 Increased speed of replication decreases the time available for DNA
repair.

α2u Globulin binding

 Best known examples:
 2,2,5-trimethylpentane in gasoline and other petroleum distillates;
 D-Limonene (fragrance);
 1,4-dichlorobenzene;

 Mode of action:
 α2u- globulin is produced in the liver by male rats at the start of
puberty;
 Passes through the renal glomerulus and then ~ 50% of the filtered
protein is reabsorbed by the tubular epithelium in the S2 segment of
the renal proximal tubule  undergoes lysosomal catabolism;

α2u Globulin binding

 Mode of action:
 Chemicals that bind to the α2u- globulin prevent its lysosomal
destruction  lysosomal accumulation in the renal proximal tubule S2
segment  lysosomal rupture  cell death  repair hyperplasia;
 A specialized example of the cytolethality mechanisms

 Only male rats have α2u- globulin therefore the mechanism is not
human relevant.

Constitutive Androgen Receptor (CAR)
mediated

 Classical examples:
 Phenobarbital in rodent liver;

 Mode of action
 Phenobarbital is the classical CAR ligand which produces CYP2B
induction, hepatocyte hypertrophy, hepatocyte hyperplasia and loss
of intercellular gap junction communication;

 At least in the case of phenobarbital, the MOA does not appear
to be relevant to humans (but this may not be the case for all CAR
agonists??)

Peroxisome proliferator activated receptor α
mediated

 Chlorinated solvents (perchloroethylene, trichloroethylene)
 Some chlorinated herbicides;
 Phthalates;
 Fibrates e.g. ciprofibrate, clofibrate;

mediated

 Mode of action
 Neoplasia is associated with tissues and organs with active fatty acid
oxidation capacity (not surprising since peroxisomes are organelles
involved with the catabolism of very long chain fatty acids, branched
chain fatty acids, D-amino acids, polyamines, and biosynthesis of
ether phospholipids);
 Neoplasia is associated with the rodent liver, rodent Leydig cells, and
pancreatic acinar cells;
 Clear species differences in PPARα response:
 Mouse and rat = high responders;
 Syrian hamster = intermediate responder;
 Primates & guinea pig = low responders.

mediated

 Mode of action
 PPARα acts as a nuclear transcription factor:

 PPARs heterodimerize with the retinoid X receptor (RXR) and bind to
specific regions on the DNA of target genes. These DNA sequences are
termed PPREs (peroxisome proliferator hormone response elements).

mediated

 Mode of action
 PPARα activation results in cell proliferation and suppression of
apoptosis: both of which are favorable to neoplasia;

 Mode of action is currently not regarded as human-relevant;
 Activation of PPARα in primates does not result in cell proliferation;
 Level of PPARαin human liver is < 10 X that of rodents;

Nongenotoxic Carcinogens: AhR agonists

 Co-planar Polyhalogenated biphenylss;
 Polyhaolgenated dibenzofurans;
 Dibenzo dioxins (TCDD);

 Mode of action;
 Appear to function predominantly as tumor promoters;
 AhR is a nuclear transcription factor;

Measuring Carcinogenesis:
Chronic toxicity studies and carcinogenesis
studies are not necessarily the same thing!

Basic, but critical, design parameters
 Number of animals, and in particular the number of surviving animals at
the take down of the study critically affects statistical power:
• 50 animals per sex, per dose at the
end of the study;
• Often means starting with 55 or 60
animals per sex per dose at the start of
the study;
• At this number of animals, the
detection above background of
cancer incidence is about 10% i.e. the
presence of rare tumor types is a very
significant finding;
• If the experimental n is less than this,
then the study will only detect very
large changes: biologically very
significant

Basic, but critical, design parameters

 For correct interpretation the following controls are necessary:
 At least one within experiment control group that is
treated/managed in exactly the same manner as all the other
groups;

 Exactly the same treatment is absolutely critical e.g. exposure
to excessive noise is a carcinogen in rodents!;

 Historical control data on spontaneous tumor incidences;

 Vehicle and/or treatment control groups may also be needed.

Maximum tolerated dose (MTD)

 Excessive premature mortality may invalidate a carcinogenesis
study and will also greatly reduce the statistical power of the
study;

 MTD = highest dose of a radiological or pharmacological
treatment that will produce the desired effect without
unacceptable toxicity;

 Generally the MTD is the highest dose in a sub-chronic study that
produces ≤ 10% body weight loss or ≤ 10% reduction in
growth/weight gain PLUS an absence of undue toxicity or deaths;



Maximum tolerated dose (MTD)

 The purpose of administering MTD is to determine whether long-
term exposure to a chemical might lead to unacceptable
adverse health effects in a population, when the level of exposure
is not sufficient to cause premature mortality due to short-term
toxic effects;

 The maximum dose is used, rather than a lower dose, to reduce
the number of test subjects in order to detect an effect that might
occur only rarely (rationale being that incidence is α to dose);

Statistical analysis

 Must take into account:

 Censored data = censoring occurs when the value of a
measurement or observation is only partially known;

 Censored data in carcinogenesis studies comes from:
 Animals that die prematurely;
 Data loss;
 Data errors;
 Animals withdrawn from the study for various reasons;

Statistical analysis

 Tumor incidence and multiplicity data is NON-PARAMETRIC count
data!
 Must use Kaplan-Meier estimator for determining survival curves
(takes into account censored data, particularly right sensored
data);
 Must talk about MEDIANS and VARIANCES rather than means
and standard deviations;
 Must use non-parametric regression techniques;
 Must use non-parametric methods for testing for statistical
differences between medians.

What defines a positive response?

 A positive response is defined as:
 The test article is associated with a statistically significant
increased tumor incidence or multiplicity compared with
acceptable within experiment negative control(s);
 The test article is associated with a statistically significant
increased tumor incidence or multiplicity compared with
relevant historical control data for that species and strain (and
animal source in some cases)
 Beware: dietary differences make BIG differences in
spontaneous tumor incidence even in the same species and
strain e.g. type of oil used in the diet can make a very big
difference;
 Housing conditions affect background tumor rates


 The carcinogenic mode of action is relevant to humans;

 The tumor type and location is relevant to humans;

 See liver, thyroid and renal slides for a discussion of the
relevance of rodent liver tumors to humans
 The interpretation tumors in rodent-specific sites such as the
rodent forestomach, zymbal gland and harderian gland, in
the absence of tumors in other locations, is always
controversial and difficult (see mode of action framework
below)

In case you were
wondering what the heck
Harderian glands and
zymbal glands are…..

No, you don’t have them.


 The presence of an increased incidence of rare tumor types in
human-relevant locations and with presumptive human-
relevant modes of action IS ALWAYS OF GREAT CONCERN!

Species and strain selection

 Under most circumstances, near lifetime exposure rat and mouse
studies are required;

 Spontaneous tumor formation varies considerably between
different strains;

 The strains with the highest background spontaneous tumor rates
are not necessarily those with the greatest response to a chemical
agent;

AN EXAMPLE OF A MODE OF ACTION
FRAMEWORK

Mode of Action of Rodent Forestomach
Tumours: Relevance to Humans.

Gross anatomy of murine forestomach after NMBA
(N-nitrosomethylbenzylamine) treatment.

Zanesi N et al. PNAS 2001;98:10250-10255

©2001 by The National Academy of Sciences

NMBA-induced histopathology of murine forestomach.

Zanesi N et al. PNAS 2001;98:10250-10255

©2001 by The National Academy of Sciences

Introduction
 Forestomach tumors/pre-neoplastic lesions in rats and mice
are a common finding in repeat-dose toxicology studies;

 Debate over the human relevance due to:
• Dose and exposure differences between rodents and
humans;
• Substantial toxicokinetic differences (exposure);
• Substantial anatomical differences;
• Substantial physiological/metabolic differences of the
forestomach epithelium;
• Different mechanisms and tumor types in humans compared
with rodents;

Dose and Exposure Problems

 Doses used in rodent oral carcinogenesis often far
exceed normal human environmental exposure conditions
(possible rare exception is some direct food additives);

 Doses that produce forestomach irritation in rodents really
should be considered as exceeding the MTD – i.e. poor
practice in rodent carcinogenesis studies and not
according to GLP/test guidelines;

Dose and Exposure Problems

 Gavage can produce forestomach irritation and is not
physiological:
 Large volumes;
 Damage to the mucosa;
 Esophageal reflux;
 Possibly replicates tablets (but not capsules);

Tissue specificity

 Forestomach carcinogens divisible into at least 3 categories:

 Produce forestomach tumors and tumors at other sites when
administered by gavage;
 Produce only forestomach tumors when administered by
gavage;
 Produce forestomach tumors and tumors when administered
by non-oral routes;

 In terms of human relevance, forestomach + tumors at other
sites is likely to be more important except in the case of site of
first contact carcinogens.

Tissue concordance/anatomical issues

 Humans do not have a forestomach or a pars esophagea:

 Roughly equivalent tissue in terms of histology is the
esophagus;
 Humans do not store food in the esophagus where as
rodents store food in the forestomach;
 Transit time through the human stomach is lower than transit
time through the rodent stomach (forestomach)  difference
in tissue exposure;
 Chemicals pass quickly through the human esophagus and
thus the exposure is very limited compared with chemical
exposure of the rodent forestomach.

Tissue concordance/anatomical issues

 Physiological issues:

 Rodent forestomach does not have a protective mucous coating 
increased tissue exposure to chemicals and more prone to irritant
effects;

 pH in rodent forestomach is higher than the pH of the human
stomach  relevant to detoxification (e.g. hexavalent chromium to
trivalent chromium in low pH of human stomach);

 Potential metabolic differences of rodent forestomach epithelium
 conversion of 2-butoxy ethanol to 2-butoxyacetic acid in rodent
forestomach but not in human stomach;

Tumour types and biology issues

 Rodents
 Predominant tumor types are papillomas (non-malignant)
and squamous cell (low malignancy – regional metastasis)
carcinomas;

 Typically located at the limiting ridge;

 Possibly have some relevance to human esophageal
squamous cell carcinoma BUT chemical exposure of the
human esophagus is much lower than in the rodent
forestomach due to much lower transit time (no storage in
esophagus);
 Not relevant to human esophageal adenocarcinoma.

Tumour Types and Biology Issues

 Humans

 All human stomach cancers are gastric
adenocarcinomas and arise from the glandular
epithelium;

 Rodent forestomach tumors have a different
histiogenesis and are not relevant to the human gastric
tumors;

Genotoxicity Issues

 Forestomach carcinogens are divisible into 2 basic groups:

 DNA reactive chemicals (classical in vivo genotoxic
carcinogens)
 Site of first contact carcinogens (generally direct acting carcinogens and
are usually highly reactive chemicals; typically direct acting alkylating
agents);
 Classical pro-carcinogen DNA reactive chemicals;

 Non-DNA reactive chemicals (classical non-genotoxic
carcinogens);
 Typically irritant chemicals or chemicals that produce local increased cell
turnover.

Genotoxicity Issuses
 Site of first contact carcinogens:
 Generally require no metabolism to be carcinogenic;
 Generally will produce tumors at other sites if the route of
administration is different  tumor location is the site of
contact;
 Generally only produce forestomach tumors in
gavage/dietary studies because of limited/no systemic
bioavailability;
 Typically alkylating agents;
 Typically genotoxicants in vitro and in vivo;

 Forestomach tumours are potentially human relevant
but only at the site of first contact in humans (e.g.
dermal exposures)

Genotoxicity Issuses
 Classical pro-carcinogen DNA reactive chemicals;
 Generally pro-carcinogens;
 Often produce tumours at more than one anatomical site
following oral dosing (at least one systemic site +
forestomach);
 Often other routes of administration also result in tumors;
 Generally systemically bioavailable;

 Human relevance of forestomach tumors depends on:
(a) was there evidence of gastric irritation; (b) were the
doses excessive (> MTD); (c) were the effects only seen
with gavage dosing/diet studies and not with drinking
water studies?

• Observation of tumours under different circumstances lends
support to the significance of the findings for animal
carcinogenicity. Significance is generally increased by the
observation of more of the following factors:

•Uncommon tumour types;
•Tumours at multiple sites;
•Tumours by more than one route of administration;
•Tumours in multiple species, strains, or both sexes;
•Progression of lesions from preneoplastic to benign to
malignant;
•Reduced latency of neoplastic lesions;
•Metastases (malignancy, severity of histopath);
•Unusual magnitude of tumour response;
•Proportion of malignant tumours;
•Dose-related increases;
•Tumor promulgation following the cessation of exposure.

Benzo(a)pyrene (IARC 1)
Parameter
Genotoxicity in vivo that is relevant to humans +
Forestomach cancers following oral dosing +
Not observed in drinking water studies, only observed with gavage/diet studies -
Only observed at doses that irritate the forestomach (> MTD) -
Uncommon tumour types; +
Tumours at multiple sites; +
Tumours by more than one route of administration; +
Tumours in multiple species, strains, or both sexes; +
Progression of lesions from preneoplastic to benign to malignant; +
Reduced latency of neoplastic lesions; +
Metastases (malignancy, severity of histopath); +
Unusual magnitude of tumour response; +
Proportion of malignant tumours; +
Dose-related increases; +
Tumour promulgation following the cessation of exposure. +

Ethyl Acrylate

•Oral gavage: dose related increases in the incidence of
squamous-cell papillomas and carcinomas of the
forestomach were observed in rats and mice. Exposure
caused gastric irritancy;

•Ethyl acrylate was tested by inhalation in the same
strains of mice and rats; no treatment-related neoplastic
lesions were observed;

•No treatment-related tumour was observed following
skin application of ethyl acrylate for lifespan to male
mice.

Ethyl acrylate (IARC 2B)
Parameter
Genotoxicity in vivo that is relevant to humans -
Not observed in drinking water studies, only observed with gavage/diet studies ?
Only observed at doses that irritate the forestomach (> MTD) +
Uncommon tumour types; -
Tumours at multiple sites; -
Tumours by more than one route of administration; -
Tumours in multiple species, strains, or both sexes; +
Progression of lesions from preneoplastic to benign to malignant; +
Reduced latency of neoplastic lesions; +
Metastases (malignancy, severity of histopath); -
Unusual magnitude of tumour response; -
Proportion of malignant tumours; -
Dose-related increases; -
Tumour promulgation following the cessation of exposure. +

Mercuric chloride (IARC 3)
Parameter
Genotoxicity in vivo that is relevant to humans -
Not observed in drinking water studies, only observed with gavage/diet ?
studies
Only observed at doses that irritate the forestomach (> MTD) +
Uncommon tumour types; -
Tumours at multiple sites; -
Tumours by more than one route of administration; (thyroid follicular cell adenomas)

Tumours in multiple species, strains, or both sexes; -
Progression of lesions from preneoplastic to benign to malignant; -
Reduced latency of neoplastic lesions; -
Metastases (malignancy, severity of histopath); -
Unusual magnitude of tumour response; -
Proportion of malignant tumours; -
Dose-related increases; -
Tumour promulgation following the cessation of exposure. -

Lost in the wilderness of mirrors?1
Don’t worry, it gets easier with time and experience.
1: From TS Eliot’s Geronation;
Famously used by JJ Angleton of CIA renown to describe the art of anti-soviet
counterintelligence.

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