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Cancer biology
Dr. med. vet. Matti Kiupel, MS, PhD, DACVP
Associate Professor, Section Chief Anatomic Pathology
Michigan State University
Diagnostic Center for Population and Animal Health
4125 Beaumont Road, Room 152A
Lansing, MI 48910, USA
Tel.: ** 517 432 2670
E-mail: [email protected]
WHAT IS CANCER?
Cancer is a group of diseases
characterized by the uncontrolled growth
and spread of abnormal cells.
Cancer Facts & Figures - 2005
American Cancer Society
www.cancer.org
WHAT IS ONCOLOGY?
Greek oncos = tumor
The study of neoplasms or neoplastic cells.
The Genetic Basis of Human Cancer, Second Edition
B. Vogelstein and K.W. Kinzler (Editors)
McGraw-Hill, New York, 2002
1
WHAT ARE NEOPLASMS?
NEOPLASIA: “NEW GROWTH”
Growth exceeds that of the normal tissues
Growth is uncoordinated
Persists in the same excessive manner after
cessation of the stimuli which evoked the change
Robbins Basic Pathology, 7th Edition
V. Kumar, R.S. Cotran, and S.L. Robbins (Eds)
Saunders, Philadelphia, 2003
WHAT ARE TUMORS?
TUMOR: “A SWELLING”
Any "lump" or 3-dimensional swelling
Described in the 1st century A.D. by Celsus as
one of the 4 cardinal signs of inflammation:
Rubor, tumor, calor, and dolor;
functio laesa (Virchow)
WHAT ARE TUMORS?
Result of a disease process in which a
single cell acquires the ability to
proliferate abnormally, resulting in an
accumulation of progeny cells.
The Genetic Basis of Human Cancer, Second Edition
B. Vogelstein and K.W. Kinzler (Editors)
McGraw-Hill, New York, 2002
2
WHAT IS CANCER?
Cancers are those tumors that have
acquired the ability to invade the
surrounding normal tissues.
The Genetic Basis of Human Cancer, Second Edition
B. Vogelstein and K.W. Kinzler (Editors)
McGraw-Hill, New York, 2002
CANCER
SIGN OF THE ZODIAC
The
Crab
Cancer in Mythology
The Second Labor
of Hercules
The Lernaian Hydra
3
WHY IS CANCER
DEPICTED AS A CRAB?
Hippocrates of Cos, 460-375 BC
coined from the Greek word meaning crab the
terms karkinos (for benign)
and karkinõma (for malignant)
to describe solid tumors.
Neoplasia. In: Anderson’s Pathology, Volume 1
G.T. Diamandopoulos and W.A. Meissner (1985)
J.M. Kissane (Editor), C.V. Mosby, St. Louis
WHY IS CANCER
DEPICTED AS A CRAB?
Galen, AD 131-201
“As a crab is furnished with claws on both sides
of the body, so in this disease the veins which
extend from the tumor represent it a figure
much like that of a crab”
In: Cancer, Volume 1
W.R. Bett (1957) Historical aspects of cancer.
R.W. Raven (Editor), Butterworth, London
WHY IS CANCER
DEPICTED AS A CRAB?
Paul of Aegina, AD 625-690
“…some say that cancer is so called because
it adheres with such obstinacy to the part it
seizes that, like the crab, it cannot be
separated from it without great difficulty...”
In: Cancer, Volume 1
W.R. Bett (1957) Historical aspects of cancer.
R.W. Raven (Editor), Butterworth, London
4
WHAT IS CANCER?
“Malignant tumors are collectively referred to as
cancers, derived from the Latin word for crab –
they adhere to any part that they seize on in an
obstinate manner, similar to a crab.”
Robbins Basic Pathology, 7th Edition
V. Kumar, R.S. Cotran, and S.L. Robbins (Eds)
Saunders, Philadelphia, 2003
TUMORS AND CANCER
Different Names for the Same Pathologic Lesion?
Neoplasm, Tumor, and Cancer
Do all of these terms refer to the same pathological
lesions?
Not all neoplasms are cancerous...
However, all cancers are considered to be “tumors”
CLASSIFICATION OF
NEOPLASMS
Hematopoietic
Leukemias
Solid tumors
Sporadic or
Hereditary
5
CLASSIFICATION OF
NEOPLASMS
Benign Tumors
Malignant Tumors
Not all neoplasms are cancerous!
CLASSIFICATION OF
NEOPLASMS
In oncology, the division of neoplasms into
benign and malignant categories is
extremely important
This classification is based on a judgement of a
neoplasm’s potential clinical behavior
Malignant tumors are collectively
known as cancers
CLASSIFICATION OF
NEOPLASMS
What characteristics of a neoplasm
suggest that it is benign versus malignant?
Cellular Features
Tumor Growth Pattern
Clinical Findings
6
BENIGN NEOPLASMS
A tumor is said to be benign when its microscopic
and gross characteristics are considered to be
relatively innocent, implying that it will remain
localized, it cannot spread to other sites, and is
generally amenable to surgical removal.
MALIGNANT NEOPLASMS
A tumor is said to be malignant when the lesion
possesses the ability to invade and destroy
adjacent structures, and spread to distant sites
(metastasize) and to cause death.
BENIGN AND MALIGNANT NEOPLASMS
The Good and The Bad?
Most people think that benign neoplasms are good,
and that malignant neoplasms are bad…
…most people are wrong!
All tumors are bad,
and some are worse than others.
7
MORBIDITY AND MORTALITY OF
BENIGN NEOPLASMS
Problems associated with benign neoplasms
depend upon several important factors:
Size of the Tumor
Location of the Tumor
Secondary Consequences
Consider several examples…
TUMORS OF THE BRAIN
Ependymoma of the brainstem
Glioma of the brainstem
Meningioma
Medulloblastoma of the brainstem
HEMANGIOMA OF THE LIVER
Hemangiomas represent benign lesions formed by endothelial cells.
These can be dangerous due to their size and their tendency to rupture.
8
BENIGN AND MALIGNANT NEOPLASMS
Distinguishing Characteristics
Cellular Differentiation
Anaplasia
Rate of Growth
Local Invasion
Metastasis
BENIGN AND MALIGNANT NEOPLASMS
Cell Differentiation and Anaplasia
Cellular differentiation of tumor cells:
• extent to which neoplastic cells resemble their normal
counterparts morphologically and functionally
Malignant neoplasms that are composed of
undifferentiated cells are considered anaplastic.
Anaplasia: - to form backward
• implies dedifferentiation or loss of the structural and
functional differentiation of normal cells
BENIGN VERSUS MALIGNANT NEOPLASMS
Benign
Malignant
General Characteristics
• Slow growth rate
• No metastatic disease
• Produces local effects
General Characteristics
• Rapid growth rate
• Metastases are common
• Can cause local and/or
distant pathophysiological
effects
9
CHARACTERISTICS OF MALIGNANT CELLS
Differentiation and Anaplasia
Examples of cellular pleomorphism in a mammary carcinoma
Pleomorphism - variation in size and shape
Stromal Response - activity in the supporting
connective tissue, “scirrhous”
CHARACTERISTICS OF MALIGNANT CELLS
Excessive and Abnormal Mitosis
BENIGN VERSUS MALIGNANT NEOPLASMS
– Each benign or malignant neoplasm will
not fit all criteria for each designation
– Some neoplasms will have features of
the opposite designation
10
TUMOR INVASION AND METASTASIS
Cancers grow by progressive infiltration,
invasion, destruction, and penetration
of the surrounding tissue.
Next to the development of distant metastases, local
invasiveness is the most reliable feature that distinguishes
malignant from benign tumors.
TUMOR GROWTH
10 12
•number of
•cancer cells
diagnostic
threshold
(1cm)
10 9
time
undetectable
cancer
detectable
cancer
limit of
clinical
detection
host
death
11
THEORETICAL TUMOR KINETICS
Tumor
kill (%)
Surviving cells Viable mass
untreated
90 (1-log)
99 (2-log)
99.9 (3-log)
99.99 (4-log)
109
108
107
106
105
Recovery of
(doubling time)
1g
100 mg
10 mg
1 mg
100 µg
3.33 days
6.66 days
9.99 days
13.30 days
3 LOG KILL, 1 LOG REGROWTH
TUMOR CELL NUMBER
Chemotherapy
Time
PROLIFERATION OF NEOPLASTIC CELLS
• Cellular proliferation
• Generation Time (T)
• Growth Fraction (G)
• Proliferation = G x 1/T
• Cellular loss
• Apoptosis
• Necrosis
12
ANALYSIS OF CELL PROLIFERATION
M
G0
Ki-67
G2
PCNA
G1
S
increasing
Mitotic Index
G1
BrdU
ANALYSIS OF CELL PROLIFERATION
CLINICAL EFFECTS
OF NEOPLASMS
Tumor Invasion and Metastasis
The Major Cause of Cancer Morbidity and Mortality
Tumor metastasis can result in major
clinical consequences and complications.
Loss of Tissue Function
Loss of Nervous Control
Pain and Discomfort
13
LOSS OF TISSUE FUNCTION
Impaired respiration due to lung metastasis
Impaired liver function due to liver metastasis
Intestinal adenocarcinoma metastatic to lung (left) and liver (right).
LOSS OF TISSUE FUNCTION
site is very important, maybe more important than size
LOSS OF NERVOUS CONTROL
Loss of voluntary motor function due to metastasis to major nerves and
loss of cognitive function due to metastasis to the brain
Histologic appearance of metastatic mammary carcinoma invasive to a peripheral
nerve (left) and metastazising to the brain (center, right).
14
PAIN AND DISCOMFORT
Related to specific sites of metastasis…
Bone Metastasis
Systemic Effects of Neoplasms
Cancer cachexia
CANCER CACHEXIA
a wasting syndrome characterized by
progressive loss of body fat and lean body mass,
accompanied by profound weakness, anorexia,
and anemia
Tumor size and extent of tumor spread generally
correlates with severity of cachexia
15
CANCER CACHEXIA
Inadequate caloric intake
Changes in energy metabolism
Increased catabolism of building blocks
(altered sensitivity to insulin)
Neoplastic cells may release substances which encourage
skeletal muscle breakdown
TNF-α ("cachectin") release from macrophages
encourages lipolysis (inhibits lipoprotein lipase)
Competion of neoplastic cells for nutrients with host cells
not a significant contributor to cachexia
PARANEOPLASTIC
SYNDROMES
symptom complexes (other than cachexia),
that cannot be readily explained by
local or distant spread of the tumor
Paraneoplastic syndromes may …
...represent the earliest manifestation of an occult neoplasm
...present significant clinical problems and can be lethal
…mimic metastatic disease, confounding treatment
ENDOCRINOPATHIES
Neoplastic cells produce hormones normal for the
cell of origin
Pituitary neoplasm - ACTH leading to Cushing’s disease
Pancreatic islet neoplasm - insulin production leading to
hypoglycemia
Sertoli cell tumor in testicle - estrogen production leading
to feminization
16
Adrenal-associated Endocrinopathy
On the adrenal
cortical cells
are LH
receptors that
are activated
after neutering
by high LH
levels
Adrenal-associated Endocrinopathy
Adrenal-associated Endocrinopathy
steroidogenic factor-1
steroidogenic acute
regulatory protein
17
Adrenal-associated Endocrinopathy
Paraneoplastic Syndromes
Paraneoplastic Syndromes
18
Paraneoplastic Syndromes
ENDOCRINOPATHIES
Neoplastic cells produce peptides which mimic a
hormone not produced by the cell of origin
“Hypercalcemia of malignancy”
Lymphoma and apocrine adenocarcinoma of the anal sac
Can lead to osteolysis and soft tissue mineralization
19
Paraneoplastic Syndromes
Paraneoplastic Syndromes
Miscellaneous: Hypertrophic Osteopathy
CANCER STATISTICS
How many people alive today have ever had cancer?
Since 1990, over 18 million new cases of
invasive cancer have been diagnosed.
9.8 million Americans with a history
of cancer were alive in January 2001…
...some cancer-free,
…others with ongoing disease.
20
COST OF CANCER
For 2004, the NIH estimated $189.8 billion…
$69.4 billion for direct medical costs
$16.9 billion indirect morbidity (lost productivity)
$103.5 billion indirect mortality costs (lost productivity)
Numbers of Cases/Year
COMPARISON OF CANCER RATES
IN HUMANS, DOGS AND CATS
4500000
Human
Dog
Cat
3500000
2500000
1500000
500000
0
All Sites
Oral Cavity Mammary
Lymphoma
WHO GETS CANCER?
In general… old people and animals
get cancer…
21
AGE-DEPENDENCE OF CANCER
30
25
Cancers at all Sites
Incidence Rate
20
15
10
5
0
<20 20-34 35-44 45-54 55-64 65-74 75-84 85+
1800
1600
1400
1200
1000
800
600
400
200
0
Cases/100,000
Total Cases (%)
35
Cancer is primarily a disease of old age…
Are there exceptions to this statement?
AGE-DEPENDENCE OF CANCER
Potential reasons:
Defective DNA repair
Decreased immunocompetence
Defective regulation of cell proliferation
Cumulative exposure to carcinogens
HEREDITARY FACTORS
The biggest single cause of
death was cancer (44%), in
particular, osteosarcomas (22%)
Boxers have a high
incidence of mast cell
tumors (among others)
The mean survival time
is 7 years, the main
cause of death is
malignant histiocytosis
22
SKIN PIGMENTATION
Solar-induced cutaneous neoplasms
Melanomas in gray horses
GENDER
Certain neoplasms are more common in one gender than the other
Hormonal dependency
Estrogen receptors in mammary neoplasia
Androgen-dependency of canine perianal gland neoplasms
and prostatic neoplasia/hyperplasia
FACTORS CONTRIBUTING TO
CANCER INCIDENCE
Genetics
Gender
Race/Ethnicity
Habitual
Exposures
Cultural
Practices
Occupational
Susceptibility to
Exposures
Cancer Development
World
Region
Environmental
Exposures
23
WHAT CAUSES CANCER?
Wrath of God?
Bad humors?
Defective immunity?
Viruses?
Other infectious agents?
WHAT CAUSES CANCER?
Circa 1550 BC
Ebers Papyrus
first major
collection of
medical
observations
made on
human
Neoplasms
The Papyrus Ebers: The Greatest Egyptian Medical Document
B. Ebbell (1937), Levin and Munksgaard, Copenhagen
WHAT CAUSES CANCER?
Hippocrates and Galen, 300 BC – 200 AD
Both regarded cancer to be a side effect of melancholia
(extreme depression characterized by tearful sadness
and irrational fears).
Cancer results from an imbalance between the black humor
(from the spleen) and the other three bodily humors (blood,
plegm, and bile).
These were the first to attribute cancer to natural causes.
24
WHAT CAUSES CANCER?
In the Dark Ages
“Cancer families,” “cancer houses,”
and “cancer villages” were described.
Environmental (infectious) causes for cancer?
Genetic predisposition to cancer development?
WHAT CAUSES CANCER?
Circa 1490-1560
George Agricola
Described 1879 as
Theophrastus Paracelsus
bronchiogenic carcinoma.
Described the “Mountain Sickness” or Bergkrankeit (cough,
chest pain, shortness of breath), that was frequently suffered
by the metal miners of Schneeberg and Joachimsthal (in the
Ore Mountains seperating Saxony and Bohemia).
WHAT CAUSES CANCER?
1775
Sir Percival Pott
Suggested a link between the high incidence of
scrotal cancer in chimney sweeps and their
exposure to “soot.”
P. Pott (1775), Chirurgical Observations Relative to the Cataract, the
Polypus of the Nose, the Cancer of the Scrotum, the Different Kinds of
Ruptures, and the Mortification of the Toes and Feet.
Hawes, Clark, and Collins, London
25
WHAT IS CARCINOGENESIS?
The pathogenic process or processes
that constitute “carcinogenesis”
represent the mechanism or
mechanisms of cancer induction.
WHAT IS A CARCINOGEN?
“Carcinogens” are agents that drive
the process of carcinogenesis.
…carcinogens cause cancer
CARCINOGENESIS MODELS
Multistage models of carcinogenesis were proposed
to explain the observation that the age-specific
incidence curves of many common cancers increase
roughly with a power of age.
Nordling, C.O. (1953) A new theory of the cancer inducing mechanism.
Br. J. Cancer 7:68-72.
Armitage, P. and Doll, R. (1954) The age distribution of cancer and
multistage theory of carcinogenesis. Br. J. Cancer 8:1-12.
26
CARCINOGENESIS IS A MULTISTAGE
PROCESS
Chemical Endogenous
Radiation
Factors
Virus
Genetic
Change
Normal
Cell
Selective
Clonal
Expansion
Initiated
Cell
Genetic
Change
Preneoplastic
Lesion
Malignant
Tumor
C.C. Harris (1991)
Chemical and physical carcinogenesis: Advances and perspectives for the 1990s.
Cancer Res. 51:5023s-5044s.
CARCINOGENESIS IS A MULTISTAGE
PROCESS
What are the various stages of carcinogenesis?
Initiation
Promotion
Conversion
Progression
What is the biological significance of each stage?
How were these stages defined?
CARCINOGENESIS IS A MULTISTAGE
PROCESS
Initiation
An rapid and typically irreversible process whereby
a carcinogen (chemical or other) produces
permanent changes in the DNA of target cells
(requires cell replication to make it permanent).
Initiating agents directly interact with cellular DNA.
(Binding of compound to DNA causes mutation)
No morphologic hallmarks of initiation
27
CARCINOGENESIS IS A MULTISTAGE
PROCESS
Promotion
The process through which tumor formation is
stimulated in tissues that have been exposed to an
initiating carcinogen (chemical or other).
Promoting agents do not directly interact with cellular DNA.
Process occurs over a long period of time (latency period)
through a series of usually reversible tissue and cellular
changes before the appearance of the first cancer cell.
Effects are cumulative (achieve a “threshold” of promotion).
CARCINOGENESIS IS A MULTISTAGE
PROCESS
Progression
The process through which the cells of a tumor
evolve in a stepwise fashion into more aggressive
forms with increasingly malignant behavior.
Concept that proliferating cells become
progressively more heterogeneous
and progressively more “neoplastic”.
Carcinogens act as initiators, promoter, or
progressors, or any combination of the above.
Initiation
Initiating
Clonal expansion
Event
2nd Critical Event
Promotion
Clonal Expansion
nth Critical Event
Clonal Expansion
Modified from Swenberg et al., 1987.
Progression
28
CARCINOGENESIS IS A MULTISTAGE
PROCESS
Initiation Promotion Conversion
Progression
Insult
Genetic
Change
Normal
Cell
Selective
Clonal
Expansion
Genetic
Change
Genetic
Change
Initiated Preneoplastic Malignant
Cell
Lesion
Tumor
Genetic
Change
Clinical
Cancer
Advanced
Clinical
Cancer
INITIATING AND PROMOTING AGENTS
IN CHEMICAL CARCINOGENESIS
Initiation alone is not sufficient for tumor formation.
Initiating agents are not complete carcinogens.
Promoting agents can induce tumors from initiated
cells, but are not carcinogenic by themselves.
Promoting agents are not complete carcinogens.
Complete carcinogens act as both initiating agent
and promoting agent.
METABOLIC ACTIVATION OF
CHEMICAL CARCINOGENS
Direct-acting carcinogens require no metabolic
activation, while indirect-acting carcinogens (or procarcinogens) require metabolic conversion to produce
the ultimate carcinogen.
Direct-acting and other ultimate carcinogens are
highly-reactive electrophiles that can react with
nucleophilic (electron-rich) sites in the cell.
e.g. benzo[a]pyrene (BP)
29
PROMOTERS OF CHEMICAL
CARCINOGENESIS
Examples of Promoting Agents
Phorbol esters, hormones, phenolic compounds, and drugs
Initiated cells respond selectively to promoting agents, while
normal cells do not respond.
Promoting agents promote cellular proliferation and
clonal expansion of initiated cells.
CARCINOGENESIS IS A MULTISTAGE
PROCESS
How were these stages defined?
Berenblum, I. and Shubik, P. (1947) A new quantitative approach to the
study of the stages of chemical carcinogenesis in the mouse’s skin.
Br. J. Cancer 1:383-391.
Dimethylbenz[a]anthracene (DMBA) and 12-O-tetra-decanonylphorbol-13-acetate (TPA)
DEFINING INITIATION AND PROMOTION
Mouse Skin Carcinogenesis Model
Initiator = no tumor
T
T
Initiator + Promotor
= tumor develops
Initiator + Delay +
Promotor = tumor
develops
Promotor = no tumor
Promotor + Initiator
= no tumor
= Initiator
= Promotor
T=
Tumor
30
EXAMPLES OF CHEMICAL
CARCINOGENESIS
1930
Specific chemicals
(dibenzanthracene)
are carcinogens in experimental
animals.
E.L. Kennaway and I. Heigler (1930)
Carcinogenic substances and their fluorescence spectra.
Br. Med. J. 1:1044-1046.
EXAMPLES OF CHEMICAL
CARCINOGENESIS
Aflatoxin B1
AFB1 is a fungal toxin that
contaminates certain food
products such as corn and
rice, and acts as a potent
hepatocarcinogen
EXAMPLES OF PHYSICAL
CARCINOGENESIS
Ultraviolet Radiation
Asbestos
UV-light exposure is strongly
correlated with development
of skin cancer (basal cell and
squamous cell carcinoma,
melanoma)
The characteristic tumor
associated with asbestos
exposure is malignant
mesothelioma of the pleural
and peritoneal cavities
31
EXTERNAL FACTORS ASSOCIATED
WITH NEOPLASIA
Hiroshima, Japan
Nagasaki, Japan
August 6, 1945
August 9, 1945
1952
Increased incidence of leukemia in Japanese survivors of the
atomic bomb blasts.
J.H. Folley, W. Borges, and T. Yamawaki (1952), Am. J. Med. 13:311-321.
Incidence of leukemia in survivors of the atomic bomb in Hiroshima and Nagasaki, Japan.
OCCUPATIONAL EXPOSURE
1974
The first cases of
angiosarcoma of the liver in
workers exposed to vinyl
chloride were described.
1929
Martland and Humphries reported a
high incidence of osteogenic
sarcomas in watch dial painters
chronically exposed to radium.
JL Creech, MN Johnson (1974)
Angiosarcoma of the liver in
manufacture of polyvinyl chloride.
J. Occup. Med. 16: 150-151.
HS Martland, RE Humphries (1929)
Osteogenic sarcoma in dial painters
using luminous paint.
Arch. Pathol. Lab. Med. 7:406-417.
Benzo[a]
pyrene
Rate Per 100,000 Population
HABITUAL EXPOSURE
80
70
60
50
Male Patients
Lung
Stomach
Prostate
Colorectum
40
30
20
10
0
1930 1940 1950 1960 1970 1980 1990
Benzo[a]pyrene is found
in tobacco smoke and is suspected to be
important in the etiology of lung cancer
Vincent van Gogh, 1886
E.L. Wynder and E.A. Graham (1950) Tobacco
smoking as a possible etiologic factor in
bronchiogenic carcinoma. A study of six hundred
and eighty-four proved cases. JAMA 143:329-336
32
Increased Numbers of Women Smokers Contributes to
the Rise in Lung Cancer Incidence Among Women
“…You’ve come a long way, baby!”
EXAMPLES OF HORMONAL
CARCINOGENESIS
Estrogens
Androgens
Hormone-dependent tumors
of the female include
mammary carcinoma and
adrenal cortical carcinoma
(and others)
Hormone-dependent tumors
of the male include perianal
gland tumors and prostatic
carcinoma (and others)
VIRAL CARCINOGENESIS
Peyton Rous produced sarcomas in chickens using a cellfree filtrate prepared from transplantable sarcoma cells.
P. Rous (1911) A sarcoma of the fowl transmissible by an agent
separable from the tumor cells. J. Exp. Med. 13:397-411.
33
RETROVIRUSES
1
2
•
•
•
1)
2)
3
3)
4
4)
5
5)
6)
include all oncogenic RNA viruses
reverse transcriptase (RNA-dependent
DNA polymerase) characteristic of group
Life cycle
Virus attaches to host cell and fuses with
membrane
Reverse Transcriptase transcribes RNA
genome to DNA
DNA copy is integrated into host cell’s
genome (provirus)
Provirus is transcribed into RNA, which is
translated into protein
Viral proteins assemble into new virion
Virion buds from infected cell
6
CONTINUUM OF RETROVIRUS-MEDIATED EVENTS
TO TRANSFORMATION OF DISEASE
Activation
Immortalization
Early Viral Effects
Polyclonal Proliferation
Transformation
Clinical
Genetic
Complications
Mutations
NOD/SCID
of ATL
Late ViralModel
Effects
Monoclonal Proliferation
Paraneoplastic Events
e.g. bone lysis,
hypercalcemia
Retrovirus Infection and Replication Dysregulation of Cell Growth Control and Altered Cell Interactions
Retrovirus Alteration of Cell Regulation and Signaling
Tumor viruses transform infected cells
•
•
•
•
•
•
Rous sarcoma virus carries src gene that
causes cell transformation
c-src plays a role in cellular processes in
normal cells, but v-src is able to transform
normal cells to tumors = oncogene
ALV can replicate without src gene
proviral DNA becomes integrated into host
DNA next to a c-src gene
provirus and c-src are co-transcribed into
hybrid RNA
after splicing out c-src introns, the hybrid
is packaged into a virus particle that may
be ancestor to RSV
How do viruses without oncogene
transform cells?
34
How do viruses without oncogene induce tumors?
•
•
•
•
•
•
slowly tranforming retroviruses activate
proto-oncogene by inserting their
genomes adjacent to cellular genes
numerous B-cell lymphomas are
produced by ALV that have their
provirus integrated into chromosomal
DNA segment that carries c-myc
proto-oncogene
integrated between first noncoding exon
of c-myc gene and the second exon
where the reading frame of c-myc begins
insertions occur randomly
transcription of c-myc through the strong
constitutively acting ALV promoter
Myc drives cell proliferation and activated
c-myc gene will multiply uncontrollably
EXAMPLES OF RETROVIRAL NEOPLASMS
Avian Leukosis-sarcoma virus
Feline Leukemia-sarcoma virus
DNA viruses
- some contain viral oncogenes
- others alter host proto-oncogenes
- Herpesviruses
- Papillomaviruses
- Poxviruses
SV40
Shope
35
Tumor viruses induced change in cell phenotype
•
•
•
•
•
•
•
•
altered morphology
loss of contact inhibition
anchorage independence
immortalization
require fewer mitogenic factors
high saturation density
unresponse to anti-growth signals
increased transport of glucose
• viral genome becomes integrated
in host cell DNA
• sucrose gradient sedimentation:
• blue = cellular DNA, green = viral
DNA, red = SV40 in cellular DNA
EXAMPLES OF DNA VIRUS NEOPLASMS
• Herpesvirus
– Marek’s disease induced
lymphosarcoma in chickens
– Lymphosarcoma in rabbits,
guinea pigs, and non-human
primates
– Lucke’s virus - renal
carcinoma in frogs
– Epstein-Barr virus
- Burkitt’s lymphoma and
nasopharyngeal
adenocarcinoma in
humans
EXAMPLES OF DNA VIRUS NEOPLASMS
• Poxviruses
• Shope fibroma virus - fibromas in rabbits
• Yaba pox - histiocytoma in rhesus monkeys
R.E. Shope (1933) Infectious papillomatosis of
rabbits. J. Exp. Med. 58:607-624.
36
EXAMPLES OF DNA VIRUS NEOPLASMS
• Papilloma viruses
• Associated with hyperplastic and neoplastic growths
• Found in a variety of species
• Equine sarcoid
Papillomaviruses
• L1 and L2 capsid proteins
– late stage structural proteins
– highly conserved
• DNA
– one strand encodes open
reading frames
– consists of 7 genes (E1-E7)
– deletion of E2 leads to
derepression of main promoter
– E6 and E7: main transforming
proteins
• repression leads to loss of
oncogenic potential
• immortalize keratinocytes
Papillomaviruses
•
Viral capsid genome
encoded late and
only in terminally
differentiated
epithelial cells
• Capsid viral antigen
and virions only
detected in
keratinized cells
• replicates in the host
nucleus
- remains episomal
(replicates
intranuclear, but
extrachromosomally)
37
Bovine Papillomaviruses
Bovine Papillomaviruses and Bracken-fern
Feline Papillomaviruses
38
Feline Papillomaviruses
Feline Papillomaviruses
GENETICS AND CANCER
Theodor Boveri
“…a particular, incorrect chromosome
combination which is the cause of abnormal
growth characteristics passed on to daughter
cells…”
T. Boveri (1914)
Zur Frage der Entstehung Maligner Tumoren
(“The Origin of Malignant Tumours”)
Gustav Fischer Verlag, Jena
39
CANCER IS A GENETIC DISEASE
DNA is the Biological Target of
Carcinogenesis
Are all carcinogens mutagens?
Are all mutagens carcinogens?
DNA and bound carcinogen (left) and chemical adduct (right).
CANCER IS A GENETIC DISEASE
Mutagens cause changes in the base
composition of the DNA
Carcinogens cause cancer
Is mutation necessary for cancer induction?
GENETIC BASIS OF NEOPLASIA
Epigenetic Mechanisms:
Alteration in gene expression
without altered DNA
Affect transcription or
translation
Genetic Mechanisms:
Direct interaction with DNA
Evidence for somatic mutation
Most carcinogens are mutagens
Chromosomal abnormalities
common in neoplasms
RESULT
Abnormal gene products
Abnormal quantities of normal gene products
“De-repression” (reactivation) of embryonic genes
40
SIMPLE GENETIC DISEASE
Simple genetic diseases are caused by inherited
mutations in a single gene that are necessary and
sufficient to determine the phenotype.
For example:
Duchenne Muscular Dystrophy
Dystrophin
Mutation
Muscular
Dystrophy
COMPLEX GENETIC DISEASE
Complex genetic diseases involve single gene
mutations that can predispose patients to pathological
conditions, but the defective gene itself is not sufficient
to guarantee development of disease.
For example:
Atherosclerosis
LDL Receptor
Mutation
Lipid
Accumulation
Atherosclerosis
CANCER AS A DISEASE STATE
“Cancer is, in essence, a genetic disease.”
Cancers become manifest following the accumulation of a critical
number of genetic (and epigenetic) alterations, in response to
imperfections in the DNA replication machinery or through DNA
damage caused by environmental mutagens.
Ultimately….
CANCER IS A DISEASE OF ABNORMAL GENE EXPRESSION.
From: B. Vogelstein and K.W. Kinzler (2002) Introduction.
In: The Genetic Basis of Human Cancer, Second Edition
(B. Vogelstein and K.W. Kinzler, Eds),
New York, McGraw-Hill, pp. 3-6.
41
CANCER
A Disease of Abnormal Gene Expression
CANCER PATHOGENESIS
“The development of cancer in humans involves a
complex succession of events that usually occur
over many decades. During this multistep process, the
genomes of incipient cancer cells acquire mutant alleles
of proto-oncogenes, tumor suppressor genes,
and other genes that control, directly or indirectly,
cell proliferation.”
W.C. Hahn and R.A. Weinberg (2002)
Rules for making human tumor cells.
New England Journal of Medicine 347:1593-1603.
WHAT ARE CANCER GENES?
“…proto-oncogenes, tumor suppressor
genes, and other genes that control, directly
or indirectly, cell proliferation.”
W.C. Hahn and R.A. Weinberg (2002)
Rules for making human tumor cells.
New England Journal of Medicine 347:1593-1603.
42
WHAT ARE CANCER GENES?
…Genes that cause cancer…
This would include classically defined…
Viral Oncogenes
Cellular Proto-oncogenes
Tumor Suppressor Genes
WHAT ARE CANCER GENES?
…Genes that contribute to cancer…
There are a number of genes and classes of
genes that contribute to neoplastic
transformation that do not conform to the
definition of classic oncogenes
or classic tumor suppressor genes.
GATEKEEPERS AND CARETAKERS
GATEKEEPERS
…Genes that cause cancer…
genes that directly regulate the growth of tumors by inhibiting
their growth or by promoting their death
CARETAKERS
…Genes that contribute to cancer…
genes that encode proteins that do not directly regulate tumor
growth, products of caretaker genes promote genetic stability
K.W. Kinzler and B. Vogelstein (2002)
Familial cancer syndromes: The role of caretakers and gatekeepers.
In: The Genetic Basis of Human Cancer, Second Edition
(B. Vogelstein and K.W. Kinzler, Eds), New York, McGraw-Hill, pp. 209-210.
43
GATEKEEPERS
Tumor Suppressor Genes
Cancers Associated
with Defects in Tumor Suppressor Genes
Retinoblastoma (Rb1)
Li-Fraumeni Syndrome (p53)
Wilms’ Tumor (WT1)
Hereditary Breast Cancer (BRCA1 and 2)
And others….
CARETAKERS
DNA Repair Genes
Hereditary Cancer Syndromes
Related to Defective DNA Repair
Xeroderma pigmentosum
Ataxia-telangiectasia
Bloom Syndrome
Hereditary Nonpolyposis Colorectal Cancer
(hMSH2, hMLH1, hPMS2, hPMS1)
GATEKEEPERS AND CARETAKERS
Normal
Gatekeeper Pathway
Caretaker Pathway
Neoplasia
How do Caretakers and Gatekeepers contribute to
carcinogenesis, individually, or in combination?
Mutation of a Mutation of 2nd Mutation of a Mutation of 2nd
caretaker
gatekeeper
caretaker
gatekeeper
gene allele
gene allele
gene allele
gene allele
leads to
leads to
genetic instability
tumor initiation
44
What is the contemporary
definition of cancer gene?
Cancer genes encode for
positive mediators and negative
mediators
of neoplastic development.
CANCER GENE CLASSIFICATION
Proto-oncogenes
Tumor Suppressor Genes
Caretakers
Gatekeepers
Positive Mediators
Negative Mediators
Cancer genes encode for positive mediators and negative
mediators of neoplastic development.
COMMON MUTATIONAL MECHANISMS
IN CANCER GENES
Chromosomal Abnormalities
Large-Scale Deletions
Amplifications
Numerical Abnormalities
Rearrangements
Sequence Alterations
Point Mutations
Insertions
Deletions
45
CHROMOSOMAL ALTERATIONS
Chromosomal alterations that represent nonlethal
genetic damage can lead to carcinogenesis.
Most chromosomal alterations
represent structural aberrations
that produce various functional
consequences.
Some chromosomal alterations
are without biological
consequence.
BIOLOGICAL CONSEQUENCES
GENE/PROTEIN ACTIVATION
Gain of gene/protein function
Loss of gene/protein regulation
GENE INACTIVATION/LOSS OF PROTEIN FUNCTION
Loss of gene expression
Loss of normal protein function
EXAMPLES
c-Ras Proto-oncogene
p53 Tumor Suppressor Gene
Functional
Capabilities
of Cancer
Cells
Hanahan and
Weinberg (2000).
Cell 100: 57-70.
46
SELF-SUFFICIENCY IN
GROWTH SIGNALS
PROTO-ONCOGENES
CARETAKERS
POSITIVE MEDIATORS
SELF-SUFFICIENCY IN
GROWTH SIGNALS
Oncogene: a cancer-causing gene of viral origin
Proto-oncogene: cellular homologue of a viral oncogene
Oncogene: any cancer-causing gene
Proto-oncogene: normal counterpart of an
activated oncogene
Positive mediators of neoplastic development include
oncogenes, as well as other cancer-promoting genes
ONCOGENES
More than 20 viral oncogenes have been identified,
each of which has a corresponding cellular
proto-oncogene
v-sis, v-raf, v-rel, v-ski, v-erbA, and others
Cellular Proto-oncogenes
Additional cellular proto-oncogenes have
been identified (without a prior viral counterpart)
More than 100 cellular proto-oncogenes
have been identified….
47
Can cancers be triggered by
the activation of endogenous
retrovirus ?
• New model in the mid 1970s explained how
tumor viruses could participate in formation
of cancer: Based on retrovirus biology
• Endogenous retroviruses can explain tumor
development in the absence of infectious
retroviral spread
• Infection of gonads can result in integration
of retroviral provirus into cell chromosomes
• Provirus becomes ensconced in genome of
descendants: endogenous retrovirus
• Activation of latent viruses can lead to
malignancy (leukemia, AKR mouse)
Human ERV
• Probing genomic DNA with DNA of infectious
retrovirus to detect ERV
• Each band in a gel channel represents a
restriction fragment in a cell genome that
carries part or all of an ERV genome
• The variability of ERV integration sites between
lab strains indicates that numerous ERV’s have
been integrated into the germ line since the
seperation from Mus musculus
• In contrast to mouse ERV genomes, human ERV
genomes detected in human DNAs show rather
similar integration sites across the species:
germ-line integration before emergence of
human species
• Polymorphic differences are largely the results of
recombination between terminal LTR sequences
at the end of individual ERV proviruses
xenotropic murine
retroviruses
Origin of endogenous retroviruses
Does this model apply to humans ?
• No tumor viruses in the majority of human cancer
• Disease-inducing retroviruses are disadvantageous and
usually eliminated, most ERV are transcriptionally silent
• No reports of infectious retroviral particles in human
tumors have been verified
• Most human ERV are relics of ancient germ-line infections
• HERV-K entered human germ line relatively recently, its
proviruses are intact, but not associated with cancer
• Thus, suspected that most of human cancer could be
induced by mutagens which mutate normal growthcontrolling genes such as cellular oncogenes
48
DNA transfection assay is a new strategy for
detecting non-viral oncogens
•
•
•
•
Do transformed cells carry mutated genes that function as oncogenes?
Introduction of DNA from chemically transformed cells into normal
cells by transfection and determination whether these recipient cells
become transformed
NIH3T3 cells (mouse fibroblasts) contact-inhibited, non-tumorigenic
Changes in cell morphology, loss of contact inhibition
Oncogenes discovered in
human cell lines are related to
those carried by transforming
retroviruses
Homology between transfected oncogenes
and retroviral oncogenes
•
DNA probe derived from H-ras
oncogene present in Harvey rat
sarcoma virus formed hybrids with
oncogene detected in NIH 3T3 cells
transformed by transfection with
DNA from bladder carcinoma cells
49
DETECTION OF FIRST HUMAN ONCOGENE
(EJ-RAS)
Tumorigenicity
Assay
High Molecular Weight
DNA From Tumor Cells
Human Bladder
Carcinoma Cells
Transfect
2-3 Weeks
Mouse 3T3 Fibroblasts
Focus of Transformed Cells
(Neoplastic?)
How are proto-oncogenes activated?
•
•
•
•
Structural
alteration or
mutation
Overexpression
Deregulated
expression
All or some of
the above
bcr-abl Translocations producing a fusion protein
• abl gene (Abelson murine leukemia virus), rapidly tumorigenic
retrovirus, maps to chromosome 9q34
• Fused with sequences clustered at 22q11, called breakpoint cluster
region = bcr (both are multidomain, multifunctional proteins)
• Identified in 95% of Chronic myelogenous leukemia (CML)
• Different breakpoints in different leukemias
50
Consequences of chromosome rearrangement
The c-abl proto-oncogene encodes a tyrosine
kinase
The Philadelphia
chromosome in CML
t(9,22)(q34;q11.1)
• The Philadelphia chromosome translocation results in activation of the
c-abl proto-oncogene through production of the bcr/abl fusion gene
• Expression of the bcr/abl gene is driven by the bcr gene promoter, and
the transcript produced represents a chimera consisting of the 5’ portion
of the bcr gene, and the 3’ portion of c-abl
• The bcr/abl protein expresses enhanced tyrosine kinase signaling
activity compared to the normal ABL protein (gain of function)
• Oncogenic activity results from inappropriate expression or
overexpression of the proto-oncogene product
H-ras point mutations
•
•
•
Point mutation by chemical
carcinogenes
Genes activated in chemically
induced tumor were identified by
DNA transfection assay
In human tumors, the activated ras
genes by single point mutations
show a mutation in codon 12, 13, 59,
and 61
A list of point mutated ras oncogenes carried by a
variety of tumor cells
H-ras oncogene activation
Identification of the transforming region of the EJ-ras gene
Normal Cells
Cancer Cells
Transfection
2-3 Weeks
2-3 Weeks
51
Identification of the transforming region of the EJ-ras gene
Normal
EJ-ras
Recombinants
DNA region responsible for transforming activity
Consequences of point mutations
Protein function of the c-H-ras
proto-oncogene product
requires interaction with GTP
• Point mutational alteration of a
proto-oncogene sequence results
in a change of amino acid in the
protein structure, typically
reflecting a nonconservative
change.
• These amino acid changes can
alter protein function.
• Oncogenic activity results from
gain of function.
• In the case of c-ras, point mutation
results in loss of GTPase activity,
resulting in constitutive activation
of the signaling function of RAS.
Detection of proto-oncogenes in increased
copy numbers in human tumor cell genomes
• Proto-oncogene amplification
• Detection of cellular protooncogenes in multiple copies in
various tumors and transformed
cell lines
• Increased expression of
amplified proto-oncogenes :
• a role in the development and
progression of these tumors
• Correlated with decreased
survival in breast cancer
Amplification of erbB2/neu in breast cancers
52
Elevation of expression of 17q genes together
with overexpression of HER2/Neu/erbB2
• Gene amplification can be
difficult to interpret
• HER2 protects cells from
apoptosis and induces growth
and division
• Amplification of HER2 occurs
as consequence of the
amplication of an entire
chromosomal segment – an
amplicon (encompasses
additional genes)
• Amplification of other genes
influences tumor phenotype
Variation on the theme: the myc oncogene
• the myc oncogene can arise by multiple
distinct mechanisms
• myc amplification in childhood
neuroblastomas by increasing copy number
• Detection of multiple copies through ISH
• Homogenous staining regions (HSRs) contain
multiple copies of N-myc encompassing gene
• broken away from normal site and
are associated with different
chromosomal regions
• High copy numbers are associated
with poor prognosis
Variation on the theme: the myc oncogene
- Chromosomal translocation of c-myc in
Burkitt’s lymphoma, placed under
control of a transcription-controlling
sequence of immunglobulin gene
- Three additional chromosomal
translocations involving chromosome 8
- Malarial parasites and EBV virus are an
etiological factor ?
- Immunosuppression predisposes to EBV?
B lymphocytes are
immortalized by EBV and
proliferate continously
Enzymatic machinery
dedicated to organizing
normal Ig gene
arrangement misfires
Inappropriate
juxtaposition of Ig gene
and c-myc oncogene
53
Oncogenic activity of Myc overexpression
• Myc belongs to family of basic helix-loophelix (bHLH) transcription factors
• act as heterodimers: Myc-Max × transcription
• Myc replaced by Mad during differentiation
• Fusion of Myc to ER > binding of ligand
(estrogen) > Myc-ER migrates into nucleus >
associate with Max and advance cell into G1/S
Cooperation Between Oncogenes
c-myc
Primary
Fibroblasts
c-ras
NIH 3T3
Primary
Fibroblasts
NIH 3T3
c-myc + c-ras
Primary
Fibroblasts
NIH 3T3
Cooperation Between Oncogenes
Mutant Ras can generate tumors in transgenic mice
% Tumor Free Mice
MMTV-Ras DNA
Myc
Ras
Myc + Ras
0
100
Age (days)
200
54
ONCOGENES
•
•
•
•
•
Normal proto-oncogene precursor is associated with cell
growth or cell death signaling
Mutated or overexpressed consistently in certain
human or animal tumor types
Mutated or overexpressed oncogene form can be
shown to augment growth signaling or cell cycle
progression when introduced into normal cells in culture
The mutated or overexpressed oncogene can transform
normal rodent cells into cancerous cells by itself or in
conjunction with another oncogene
The oncogene can cause tumors in mice when
overexpressed as a transgene
POSITIVE MEDIATORS
OF NEOPLASIA
Neoplastic disease is characterized by
uncontrolled cell proliferation
Positive mediators of neoplastic development function to
promote cell cycle progression and cell proliferation
POSITIVE MEDIATORS
OF NEOPLASIA
Positive Growth Factors
EGF, TGF-α, PDGF-β, and others
Growth Factor Receptors
EGFR, PDGFR, C-KIT and others
Signal Transduction
K-RAS, BRAF, β-catenine
Nuclear and Cell Cycle Regulation
C-MYC, CYCLIN D and E, CDK4
55
FUNCTIONAL SUBCELLULAR LOCALIZATION
OF PROTO-ONCOGENE PRODUCTS
Growth
• Highly conserved in evolution Growth Factors
Factors
Receptors
• Important regulators of normal
cell growth and differentiation
GTPase
• Maintain the ordered
Proteins
progression of the cell
Cytoplasmic
Serine-Threonine
cycle, cell division, and
Guanine
Kinases
Nucleotide
differentiation (lost in cancer)
Exchange
• Mutation that alter the
Proteins
structure, levels, or sites of
Nuclear
expression of the gene
Transcription
Factors
products > activation of
Cytoplasmic
Membrane-associated
oncogenic potential
Tyrosine Kinases
Growth signal transduction
• Most proto-oncogene products
are components of growth
signal transduction pathways.
• The signal often begins at the
cell surface, ends in the
nucleus, and results in cell cycle
entry and progression.
• The presence of shaded
components in the pathway
represent proteins that can be
mutated to become oncogenic.
• Once oncogenic, such proteins
can promote constitutive
signaling to downstream
components independent of
upstream stimuli.
Oncogene activated deregulated signaling
Cell surface
Growth
Signal
+
Upstream
Effector
+
ProtoOncogene
Protein
+
Downstream
Effector
Regulated Cell Division
Normal Regulated Growth Signaling
No Cell
Surface
Growth
Signal
Oncogene Activated Deregulated Signaling
Oncogene
Protein
+
Downstream
Effector
Continuous Cell Division
56
Growth factor receptors
There are a large number of growth factor receptors on the surface of the cell that mediate
the initial phase of growth signal transduction, usually through the binding of a growth
factor ligand. These receptors can be grouped into two major categories: those with
intrinsic tyrosine kinase activity and those without. Many of the receptor tyrosine kinases
(RTKs) can be oncogenes. RTKs implicated in cancers are indicated in bold and italics.
Ligand
binding
membrane
Tyrosine
kinase
Receptor tyrosine kinases
The key structural domains of receptor tyrosine kinases are:
1. Extracellular ligand binding domain
2. Transmembrane domain
3. Cytoplasmic tyrosine kinase domain - highly conserved
4. Cytoplasmic kinase regulatory domains - usually C terminal
Structure of EGFR receptor
Receptor tyrosine kinases
Y
Serine
Threonine
Kinase
P T
P S
The conservation of the
tyrosine kinase domain
and its presence in so
many of the oncogenic
receptors indicate that it
is a critical component of
the growth signaling
response.
Kinase
Target
Protein
Y P
Y S T
P P P
Dual
Specificity
Kinase
Phosphorylation of proteins on either
tyrosine, serine, or threonine is very
common.
T
S
Tyrosine
Kinase
The Human Genome Project identified about 520
protein kinases. About 90 are tyrosine kinases.
Yet tyrosine kinases are disproportionately
represented among the oncogenes.
Phosphorylation by a kinase can induce:
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Conformational changes
Dissociation of other interacting proteins
Reassociation of other interacting proteins
Reassociation or dissociation with RNA or DNA
Induction of biochemical or enzymatic activity
Reduction of biochemical or enzymatic activity
Change in cellular localization
Initiation or termination of biological processes
(e.g. apoptosis, cell cycle progression, etc.)
57
Receptor monomers and dimers
Inactive receptor monomers are in equilibrium with inactive and
active receptor dimers
Binding of growth factor ligand may induce a dimerization of the
bound receptors, resulting in stabilization of active dimer formation
and an activation of the cytoplasmic tyrosine kinase
•
•
Inactive disulfide bridged insulin-receptor
dimers are in equilibrium with active
dimers. Insulin binding stabilizes the
active dimeric state leading to PTK
activation.
Variations in receptor dimerization
A.
B.
C.
VEGF binds as a dimer to two monomers of its receptor Flt-1 thereby
bringing them together
each of the FGF-2 ligands binds to the ligand binding domain of a
receptor monomer FGF-R1; each ligand-binding domain of the receptor
subunits is composed of 2 subdomains; receptor dimerization occurs
only if the 2 FGF ligand molecules are bound to a heparin molecule (a
glycosaminoglycan component of the extracellular matrix)
molecules of TGF-α bind individually to the 2 subunits of EGF-R
Growth factor receptor homo- and
heterodimerization
While the EGF receptor can homodimerize after binding to EGF ligand,
it can also form heterodimers with three other members of a family of
EGF-like receptors (EGFR/HER/erbB). The ligand specificity (where
known) is indicated for each family member. Interestingly, HER3 does
not have an active
tyrosine kinase domain
and must heterodimerize
ErbB2
ErbB3
ErbB4
to form an active
signaling complex.
the erbB receptor consists of three
domains: a ligand-binding
extracellular domain containing two
cysteine-rich regions (CR1 and
CR2), a transmembrane domain, and
an intracellular domain containing a
tyrosine kinase region.
Note at left that homodimers of the ErbB/HER family exhibit weaker
signaling than do heterodimers, particularly heterodimers
Inactive with ErbB2.
This has major significance for human breast cancers
kinase where ErbB2 is
often amplified and overexpressed. Note also the many biological
effects that result from heterodimer signaling.
58
Growth factor receptor homo- and
heterodimerization
Homodimers of the ErbB/HER
family exhibit weaker
signaling than do
heterodimers, particularly
heterodimers with ErbB2.
This has major significance
for human breast cancers
where ErbB2 is often amplified
and overexpressed.
Interactions of ErbB receptors
The monomeric structure of ErbB2
(with no ligand) resembles the activated
dimer state (with ligand) of the EGF
receptor (ErbB1). Thus, ErbB2 is
always in a ready state to form dimers
without the usual ligand-dependent
conformational shifts necessary for the
ligand binding family members.
Some ErbB family members,
such as ErbB2, inefficiently
form homodimers and may be
dependent on heterodimerization
with ErbB3. Once the ErbB2/
ErbB3 heterodimer is formed,
signaling is much more
efficiently initiated
Dimerization of receptors induces tyrosine
autophosphorylation
Tyrosine autophosphorylation following ligand binding and dimerization
- the conformational changes and dimerization of the ligand bound
receptor juxtapose the cytoplasmic tyrosine kinase domains and result
in trans-phosphorylation of the two monomer cytoplasmic domains.
- these phosphorylated
tyrosines provide
docking sites for
downstream signaling
targets containing
PTB or SH2 domains.
59
Deregulated firing of growth factor receptors
- deletion of extracellular domain results in truncated receptor
- can be the result of mutation in receptor-encoding gene or alternative
splicing of receptor pre-mRNA
- as a result receptor emits signal into cell without ligand binding
- EGF receptor is capitated in one third of glioblastomas
Gene fusion causes constitutively dimerized receptors
•
•
•
receptor dimerization occurs
in a number of malignant
tumors when the genes
encoding growth factors
become fused to unrelated
genes that happen to specify
proteins that normally dimerize
or form higher order oligomers
as a consequence the receptor
portion of these hybrid fusion
proteins are dragged together
by the oligomerizing proteins
to which they have been joined
the end result is a constitutive
ligand-independent
dimerization of the affected
receptors
c-kit proto-oncogene
L
M
N NC
60
Kit receptor activation
• juxtamembrane domain (JM) sits above the N-terminal lobe of the
two-lobed Kit tyrosine kinase
• ligand binding (SCF) to receptor results in dimerization and
transphosphorylation of tyrosine residues in JM domain, thereby
dissociating it from N-terminal lobe (“moves out of the way”)
• results in transphosphorylation of normally obstructing tyrosine
residue in catalytic cleft, contributes to kinase activation
TGF-β receptor
• the TGF-β has a structure that is similar to receptor tyrosine kinases
• both signal through cytoplasmic kinase domains
• kinase domains of TGF-β receptors phosphorylate serine and
threonine, rather than tyrosine residues
• type II receptor is brought in contact with type I receptor through TGF-β
• phosphorylates and activates kinase carried by type 1 receptor
Cytokine receptor
• the interferon receptor (IFN-R) carries noncovalently
attached tyrosine kinases of the Jak family (Tyk2, Jak1)
• upon binding of α-interferon the 2 tyrosine kinases
transphosphorylate and activate each other
• subsequently they phosphorylate the C-terminal tails of
the receptor subunits, thereby inducing the signaling
61
Notch receptor
• embodies very primitive type of
signaling
• after binding ligand, delta, Notch
undergoes two successive
proteolytic cleavage events
• resulting C-terminal fragment is
freed and migrates to the nucleus
• Notch pathway contributes to
Ras-mediated cell transformation
• constitutively active mutant
forms of Notch fire in ligandindependent fashion and have
been found in 50% of adult T-cell
leukemia
Patch-Smoothened signaling system
• hedgehog pathway utilizes its
own unique signaling system
• Smoothened, a seven-membranespanning surface receptor is
normally inhibited by Patched,
which contains a 12 membranespanning domains
• Gli is cleaved into a protein that
moves into nucleus functioning
as a repressor of transcription
• when the hedgehog ligand binds
to Patched, it releases
Smoothened, which prevents
cleavage of Gli so it can act as an
inducer of transcription
Mutant forms of
Ptc and Smo in
basal cell
carcinomas
Frizzled receptors signaling
• receptors of the Wnt proteins
are all members of the Frizzled
family of transmembrane
proteins
• with no ligand, Axin and Apc
allow glycogen synthase
kinase-3β (GSK-3β) to
phosphorylate β-catenin
• this marks β-catenin for rapid
destruction
• when wnt binds to Frizzled,
the activated receptor via the
Dishevelled protein causes
inhibition of GSK-3β
Tethered to ECM
62
Serpentine receptors
• seven-membrane-spanning receptors,
serpentine, are associated with
heterotrimeric G proteins (α, β, γ subunit)
• binding of a ligand, stimulates α subunit
to release GDP and bind GTP instead
• the α subunit dissociates from the βand γ-subunits
• independently, the α as well as the
β + γ complex can regulate enzymes that
evoke a variety of downstream signals
• the signal is halted after the α-subunit
hydrolyzes its bound GTP and
re-associates with other units
• very common receptor that rarely
contributes to cancer
Integrine receptors
• important for growing anchorage dependent
• heterodimeric, with α- and β-subunit
• associated with ECM through ectodomains
and cytoplasm through cytoplasmic domains
• β-subunit links to cytoskeleton (actin, talin,
vinculin), send signals “inside-out”
Integrine receptors
• inactivation of β-integrin in
mouse mammary tissues
• minimal effect on mammary
development in controls (A) and
knock-outs (B) [β-integrin = red]
• transgenic mice (expressing
polyomavirus oncogene) prone
to mammary tumorigenesis
• abundance of pre-malignant
hyperplastic nodules (black)
versus normal epithelium (red)
in β-integrin positive mice
• reduction of premalignant foci
in knock-out mice (survive, but
can’t proliferate)
63
Alternative mechanisms of transformation by Ras
•
•
•
•
•
•
ras-transformed cells release
growth factors (e.g. TGF-α)
act in autocrine fashion (activate
EGFR) promoting proliferation
co-expression of TGF- α and EGFR
in a breast carcinoma
“normally” Ras acts “upstream” of
the EGF receptor
alternatively, Ras can be in the
middle of signaling cascade,
operating “downstream” of EGFR
How is Ras activated “downstream”?
Regulation of RAS signaling
GTP
Operate as binary
switches similar
to heterodimeric
G proteins
GTP
Active
Ras
Downstream Signals
(Raf1, MAPK,
MAPKK, RSK, c-jun)
GTP
Upstream Signals
GDP
Guanine nucleotide
exchange factors (GEF)
Inactive
Ras
GDP
GTPase-activating
Proteins (GAP)
Functional Consequences of c-H-ras
Proto-oncogene Activation
GTP
GTP
Active
Ras
Downstream Signals
(Raf1, MAPK,
MAPKK, RSK, c-jun)
GTP
Upstream Signals
GDP
Guanine nucleotide
exchange factor (GEF)
Inactive
Ras
GDP
oncogenic
mutation
GTPase-activating
Proteins (GAP)
64
Cytoplasmic Signaling
•
•
responding to mitogenic
signals, growth factors can
release a diverse array of
biochemical signals
transcription of some
genes is observed within
minutes (immediate early
genes) while transcription
of others (delayed early
genes) occurs with a lag
since they require
synthesis (can be blocked
by cycloheximidine)
Tyrosine phosphorylation and SH2 domains
•
•
•
•
•
•
Src has 3 homology domains:
SH1 harbors the catalytic function
SH2 acts as an intracellular
receptor for specific
phosphotyrosines whose unique
identities are determined by the
oligonucleotide sequence on their
C’ side
SH3 recognizes and binds certain
proline-rich domains of substrate
as a consequence of ligandinduced transphosphorylation, a
RTK displays on its cytoplasmic tail
an array of phosphotyrosines
phosphotyrosines will attract and
bind to specifc SH2 domain
resulting in downstream signaling
non-receptor tyrosine kinase Src
•
•
•
•
the SH2 group of Src normally binds intramolecular to phosphotyrosine
residue at position 527 of the C-terminus
this obstructs catalytic cleft (between N- and C-lobe), N-lobe bound to SH3
when activator (PDGF-R) becomes phosphorylated, there is a switch from
intramolecular to intermolecular binding (SH2 binds to PDGF-R)
SH3 detaches and catalytic cleft is opened for secondary phosphorylation
SH3
SH2
SH3
SH2
65
Activation of Ras through SH2 groups
•
•
•
•
an association of Sos, the Ras
guanine nucleotide exchange
factor (GEF) with ligandactivated growth factors is
achieved by bridging proteins
2 SH3 domains of Grb2 bind to
prolin-rich domain of Sos
while its SH2 domain binds to
phosphotyrosine located on:
- C-terminal tail of receptor
- Shc, which binds to receptor
anchoring Sos to receptor will
induce release of GDP from
Ras thereby activating it
this mechanism explains how
growth factors acquire
signaling specificity
Bridging proteins Grb2 and Shc
Jak-STAT pathway
•
•
•
•
pathway depends on the actions of Jak tyrosine kinases
Tyk2 and Jak1, which are attached noncovalently to a
number of cytokine receptors (interferon, TPO, EPO)
following binding of ligand the 2 tyrosine kinases
transphosphorylate and activate each other and
subsequently they phosphorylate the C-terminal tails of
the receptors, which than attract STAT proteins that bind
through their SH2 domain and become phosphorylated
STATs dimerize and translocate to nucleus
constitutively activated in many cancers (eg melanomas)
The Ras-Raf-MAP kinase pathway
•
•
•
signaling cascade of overall plan MAPKKK > MAPKK > MAPK
most important pathway for transforming powers of Ras oncogene
stimulates expression of important growth-regulating genes
mutated in 60-70%
of melanomas
MAPK = mitogen activated kinase
activation of promoters of the two
immediate early genes encoding the
Fos and Jun transciption factors
66
The Ras-PI3 kinase pathway
•
•
•
Ras can associate with and activate phosphatidylinositol 3-kinase (PI3K)
results in formation of phosphatidylinositol (3,4,5)-triphosphate (PIP3) and
thereby activation of Akt/PKB and Rho-GEFs
Akt/PKB can suppress apoptosis by blocking Bad and stimulate cell
proliferation by blocking GSK-3β (antagonist to cyclin D1 and Myc)
The Ras-Ral pathway
•
•
•
interactions of ras with Ral-GEF activate the Ral proteins Ral-A and Ral-B
GTP-bound ral proteins proceed to activate numerous downstream pathways
Cdc42 and Rac enable motility through their effects on cytoskeleton
Complexity of the Ras effector pathways
67
INSENSITIVITY TO
ANTI-GROWTH SIGNALS
TUMOR SUPPRESSOR GENES
GATEKEEPERS
NEGATIVE MEDIATORS
INSENSITIVITY TO
ANTI-GROWTH SIGNALS
About 20 tumor suppressor genes have been
identified…
Genetic Linkage Analysis
Loss of Heterozygosity (Allelic Deletion)
Mutation Analysis
Candidate Genes
Rb1, WT1, p53, APC, NF-1, NF-2, BRCA1, BRCA2,
VHL, p16, DCC, and others
TUMOR SUPPRESSOR GENES
How did we identify oncogenes?
– Identification of retroviral oncogenes
– Molecular cloning of oncogenic DNA
sequences at chromosomal breakpoints
– DNA tranfection assay with DNA derived from
cancers
• Identification of the TSG is far more difficult.
•
Why ?
68
EVIDENCE OF TUMOR SUPPRESSOR GENES
Somatic cell hybrids
generated from
tumorigenic and
nontumorigenic
cell lines (fusion
with inactivated
Normal Cell
Sendai virus) are
(Fibroblast)
typically not
tumorigenic,
suggesting that
specific chromosomes
confer tumor suppression
Cell
Fusion
Tumor Cell
Nontumorigenic Cell
EVIDENCE OF TUMOR SUPPRESSOR GENES
• Wildtype genes antagonize
cancer phenotypes > cancer
phenotype is “recessive”.
• Recessive means loss of
both alleles of TSG
• Loss of tumor suppressor Virus induced
protein function by
mutation is lot more easier
than hyperactive mutation
of oncogenes by mutation
• Case against TSG:
probability to inactivate a
single gene is 10-6 per cell
generation, so 10-12 per
cell generation for
silencing both copies
Non virus induced
PEDIATRIC RETINOBLASTOMA
• Retinoblastoma is the most common
intraocular malignancy in children
(worldwide incidence between 1 in 13,500
and 1 in 25,000 live births)
• Tumor arises from an oligopotential stem
cell precursor of multiple retinal cell types
• Differences between
unilateral versus
bilateral retinoblastoma
early versus late onset
• Why?
69
Unilateral versus bilateral retinoblastoma
•
•
•
•
Bilateral Retinblastoma:
– other types of cancer (osteosarcoma,
Ewing sarcoma, leukemia, lymphoma)
Unilateral Retinoblastoma: no other tumors
Sporadic and hereditary tumors - why?
Pedigree shows multiple generations of a
kindred afflicted with familial retinoblastomas
– confirmation of involvement of genetic
alteration by cytogenetic analysis
– microscopically visible deletion of one
chromosome 13, band q14
– autosomal dominant hereditary form of
retinoblastoma : large deletions of chromosome 13
– germ-line mutation in hereditary retinoblastoma
– usually of parental lineage (more cell divisions in sperm)
Unilateral versus bilateral retinoblastoma
• The rate of familiar
tumors was consistent
with a single random
event, while the sporadic
tumors behaved as if two
random events were
required for their
formation
• Predicted that one of the
alleles of the Rb genes in
familial Rb carries
mutant form
10-6 /cell division
10-12 /cell division
• Inherited and somatic changes may collaborate in tumor formation
• Linked the notion of recessive genetic determinants for human
cancer to the somatic cell genetic studies
TWO-HIT HYPOTHESIS FOR
RETINOBLASTOMA
Female
The two-mutation model accounts
for the dominant inheritance of
susceptibility to retinoblastoma
However, it was recognized
that the susceptibility
gene did not function as a
single dominant determinant
of neoplastic transformation
at the cellular level
Normal
Male
Affected
A.G. Knudson, Jr. (1971)
Mutation and cancer: Statistical study of retinoblastoma.
Proc. Natl. Acad. Sci. U.S.A. 68:820-823
70
HEREDITARY RETINOBLASTOMA
Rbmut
Sperm
Rb
Rbmut Rbmut
Egg
Homozygous Mutant
Tissue
Rbmut Rb
Heterozygous
Individual
Somatic
Mutation
Development of
Retinoblastoma
(usually with multiple tumors)
SPONTANEOUS RETINOBLASTOMA
Rb Rb
Rbmut Rbmut
Homozygous Normal
Individual
Homozygous Mutant
Tissue
First Somatic Mutation Second
Somatic Mutation
Rbmut Rb
Heterozygous Tissue
Development of
Retinoblastoma
(usually with a solitary tumor)
MECHANISMS FOR
INACTIVATION OF 2ND ALLELE
Rbmut Rb
Heterozygous Tissue
Chromosome
Loss
Rbmut
Hemizygous Mutant
Mitotic
Recombination
Chromosome
Duplication
Second
Mutation
Rbmut Rbmut
Homozygous Mutant
71
MECHANISMS FOR
INACTIVATION OF 2ND ALLELE
- Mitotic recombination can
lead to LOH of Rb gene
- Genetic material is exchanged
between two homologous
chromosomes through the
process of genetic crossing
over (G2 or M phase)
- Homo- or heterozygous daughter cells
•
•
•
•
Gene conversion
DNA polymerases initially begin to use the
strand on one chromosome as a template
for synthesis of daughter strand
May continue replication by jumping to
homologous chromosome and jump back
A mutant TSG, like mutant allele of Rb, may
be transmitted from one chromosome to its
homolog, replacing the wild type
Loss of heterozygosity
- The Rb gene often
undergoes LOH in tumors
- Study of chromosome
metaphase spreads
- Second gene lying in 13q14
chromosomal region encodes
enzyme esterase D
- LOH affects whole region
- Isoforms seperated by gel
electrophoresis
Loss of heterozygosity
- The loss of genes localized
to the region of LOH could
result in haploinsufficiency
or unmasking of the
functional expression
of a recessive or deficient
allele.
72
Approaches to identify TSGs
•
•
Cytogenetic studies to identify constitutional chromosomal
alterations in cancer patients; deletion of specific chromosome or
specific region: difficult due to small portion of cancer cells
– Familial retinoblastoma; Ch13q14 in blood sample
– Wilms tumor; 11q13
– adenomatous intestinal polyps; 5q
Linkage analysis; trace the genetic markers from implicated
chromosomal region co-segregated with the inheritance of the
disease phenotypes; pinpoint the location of the TSG
–
•
•
LOH studies
– Retinoblastoma > decreased levels of esterase D
Positional cloning of the TSGs
Monochromosome transfer
Chromosome transfer
Transfer of Human
Chromosome 11
Suppression
G401 Wilms Tumor Cells
Transfer of Human
Chromosome 13
Transfer of Human
Chromosome X
Tumorigenic
Tumorigenic
Chromosome transfer
Transfer of Human
Chromosome 11
Suppression
G401 Wilms Tumor Cells
Transfer of Chromosome
11del(p13)
Transfer of Chromosome
11del(p15.5-p14.1)
Suppression
Tumorigenic
73
TSG inactivation by promoter methylation
•
•
•
•
•
Methylation of cytosine groups located in a position that is 5” to
guanosines, in the sequence CpG > can alter DNA covalently
In vicinity of promoter can repress transcription of gene
Histone deacetylase enzyme complexes bind to Me-CpG, remove
acetate groups and convert chromatin to non-transcriptive state
Newly synthesized daughter strands are methylated by
maintenance methylases > methylation state is heritable
DNA methylation does not altered nucleotide sequence: epigenetic
Methylation-specific PCR (MSP) to gauge
methylation status of promoters
- Bisulfite treatment causes
conversion of cytosin to uracil
- Methyl-cytosine is resistant
- Primers will anneal to
U-containing DNA recognize
it as T
- Non-methylated primers will
fail to properly anneal to CpG
that was methylated
- Amplification of DNA
fragments whose presence
indicates methylation or
absence thereof
U behaves like T
Methylation of the RASSF1A promoter
•
•
•
•
•
•
•
•
•
•
Bisulfite sequencing technique to determine methylation of the
CpG island in which the promoter of the RASSF1A tumor
suppressor gene is embedded
Each circle indicates the site of a distinct CpG dinucleotide
Location of CpGs in promoter is indicated by map
Blue circles indicate that CpG was methylated
Open circle are unmethylated
Analysis of 5 DNA samples
Almost all CpG sites in RASSF1A
CpG island are methylated
Adjacent tissue contains some
cells that are methylated
No methylation in control tissue
TSG suppression by
hypermethylation
74
Methylation of the p16INK4A promoter
•
•
•
In-situ hybridization to determine methylation status of DNA
Low and high grade cervical intraepithelial squamous lesions
with varying degree of methylation
Normal surface epithelium (arrow) is non-methylated
Methylation of promoters of multiple
TSG within tumor cell genomes
INACTIVATION OF TUMOR
SUPPRESSOR GENES
Loss of Protein Function
Small Sequence Mutations
Missense mutants result in defective protein
Insertion mutations result in defective protein
Chromosomal Alterations
Gene deletion results in loss of expression
Gene rearrangement results in defective protein
Epigenetic Regulation
Overexpression of negative regulator inhibits protein
function
75
TUMOR SUPPRESSOR GENES
APC - Familial Adenomatous Polyposis (FAP)
BRCA1 - Breast and Ovarian Carcinoma
BRCA2 - Breast Carcinoma
p53 - Breast, Colon, and Lung Carcinomas, (and others)
Rb1 - Retinoblastoma, Osteosarcoma, Breast, Bladder,
and Lung Carcinomas
NORMAL FUNCTIONS OF TUMOR
SUPPRESSOR GENES
Regulation cell cycle progression
Cellular differentiation
Cell adhesion and cell structure
Cell signaling
Regulation of gene expression
DNA replication and repair
And others?
NF1 (Neurofibromatosis type 1)
• A common autosomal
dominant disoder(1/3,500)
• Also develop glioblastoma,
pheochromocytoma, and
myelogenous leukemia
• Show Café au lait spots:
hypergimentation, Lisch
nodules, distinctive boney
lesions etc
• Chromsome 17q11.2
• Genetic behavior parallels
Rb1 > LOH
• Encodes neurofibromin
• Functions as GTPase
activating protein (GAP)
76
NF1 acts as an negative regulator of Ras signaling
• Function as Ras-GAP protein
• After growth factor stimuli, NF1
rapidly is degraded, but NF1 levels
return back to normal to shut
down further the Ras-signaling:
Negative feedback
• Cells lacking NF1 function have
elevated levels of activated Ras
• NF1+/- cells have elevated levels
of Ras-GTP > substantially
increased levels of Ras-signaling
• Not both alleles have to be lost
• “Haploinsufficiency” : p27, smad4,
Pten etc, but not Rb, p53
Haploinsufficiency
•
•
•
•
•
•
•
Definition: a single wild type allele is unable to
produce enough transcription product for normal
cellular function
A single mutant allele of a tumor suppressor
poses an intermediate risk for cancer
Not all mutant alleles are null recessives (alleles
incapable of producing functional product)
Hypomorphic recessive mutations: reduced but
not absent functionality
Dominant negative mutations: nonfunctional
product that directly interferes with the function
of the normal protein
Pleiotrophy: functioning of one gene in multiple
cellular processes. Some functions maybe more
dosage dependant than others
Extragenic modifiers: can both enhance and
suppress the ability of tumor suppressors to
prevent cancer
Classic tumor suppressor genetics
inherited cancer
sporadic cancer
Haploinsufficient tumor suppressor
genetics
Von Hippel - Lindau diseases: pVHL
modulates the hypoxic response
•
•
hypoxia inducible transcription factor-1 (HIF-1) is synthesized at a high rate
under normoxia and immediately degraded by the actions of pVHL: proline
hydroxylase oxydizes 2 prolin residues of HIF-1, which enables pVHL binding
and ubiquitylation of HIF-1
under hypoxic conditions HIF-1 escapes ubiquitylation and activates VGEF
77
Cell cycle clock is the master governor to
decide proliferation or not
• Molecular circuitry operating in
the cell nucleus that processes
and integrates a variety of
afferent signals originating from
outside and inside the cell and
decides whether or not the cell
should enter into the active cell
cycle or retreat into a
nonproliferating state.
• If cell proliferates, circuitry
proceeds to program the
complex sequence of
biochemical changes that
enable cell to divide into two
daughter cells.
Cell cycle regulation is based on cyclically
activated serine/threonine protein kinases
Changing levels/availability of cyclins > activate catalytic function of CDKs
D type cyclins convey information about extracellular mitogenic signals
Other cyclins operate on preordained schedule (move cycle only forward)
Checkpoint controls ensure correct progression through cell cycle
Cyclin-CDK complexes are also regulated
by CDK inhibitors (p15, 16, 18, 19, 21, 27, 57)
78
Control of cell cycle by mitogenic signals
• TGF-β induces p15INK4A (blocks CDK4/6) and weakly p21Cip1 expression
• Akt/PKB phosphorylates p21Cip1 (nucleus) and p27Kip1 (cytoplasm)
• phosph. p21Cip1 goes to cytoplasm, can’t inhibit cyclin-CDK complexes
• phosph. p27Kip1 can’t go to nucleus to inhibit cyclin-CDK complexes
• without active CDK4/6 cell can’t advance to R point, cell blocked in G1
• other cyclin-CDK complexes are active through remainder of cell cycle
Inhibitory signal
Stimulatory signal
DNA damage
p15INK4A
Suppression of p27Kip1 function by Akt/PKB
• various breast cancers, like breast carcinoma, show different p27Kip1
locations that reflect activation state of kinases Akt/PKB
• phosphorylated p27Kip1 can’t go to nucleus to inhibits cyclin-CDK
• cytoplasmic localization of p27Kip1 associated with decreased survival
N = nuclear
N+C = nuclear + cytoplasmic
Cell cycle phosphorylation of Rb
is the determinant of R point
• Phosphorylation of Rb is
closely coordinated with
cell cycle advance
• Cyclin D-CDK4/6 complex
hypophosphorylates Rb
• CyclinE-CDK2 complex
hyperphosphorylates Rb
(Active Rb)
(Inactive Rb)
79
E2F transcription factor enables Rb checkpoint
• E2F/DP1/RB complex represses gene transcription
• hyperphosphorylation of Rb at R point activates E2F transcriptional activity
• E2F is inactivated and degraded in S phase
RESTING CELL
Passage through
the G1 restriction
point
Activation of E2F
transcriptional
activity
Transcription of
cell cycle genes
(Cyclins A, E, CDK1)
DNA replication genes
Irreversibility of cell cycle progression
• cyclin E-CDK2 complex drives Rb hyperphosphorylation > release of E2F
from Rb control > synthesis of cyclin E: positive feedback loop
• activation of small number of cyclin E-CDK2 > phosphorylation of p27Kip1 >
marked for ubiquilation and degradation > liberation of more cyclin E-CDK2
Counterbalancing controls on cyclin D1 levels
• mitogenic signals influencing cyclin D1:
• 3 Ras signaling pathways:
• AP-1 transcription factor (Fos/Jun)
acts on cyclin D1 promoter > ×
• activates PI3 kinase and thus Akt/PKB
> inhibits actions of GSK-3β > this
spares β-catenin from phosphorylation,
ubiquilation and degradation > β-cat
partners with Tcf to induce cyclin D1
• activates Erk > opposite effect >
phosphorylation of cyclin D1 > marked
for ubiqilation and degradation
• growth factors > growth factor receptors >
Ras > cyclin D1 and E > inactivation of pRb
> activation of E2F > S-phase entrance
G1-S progression
80
Myc oncoprotein deregulates control of cell cycle progression
• Myc modulates a number of positive and
negative regulators of cell cycle advance
• the heterodimer Myc-Max induces growthpromoting proteins cyclin D2 and CDK4 >
hypophosphorylation of pRb > advance
through G1
• Myc-Max increases expression of Cul1,
which degrades the p27Kip1 CDK inhibitor
liberating cyclin E-CDK2 complexes from
inhibition
• by association with second transcription
factor, Miz-1, Myc can also act as
transcription repressor: - represses
expression of p15INK4B and p21Cip1 (CDK
inhibitors that shut down CDK4/6 and
CDK2), counteracts TGF-β
• Myc can induce expression of genes
encoding E2F 1, 2 and 3 > Ø pRb
How TGF-β blocks the cell cycle progression
• TGF-β “last word” in determining
whether or not a cell proliferates
• TGF-β through receptor phopshorylates
Smad proteins, such as Smad 3
• Smad 3 and 4 form heterodimer
• migrate to the nucleus
• team up with Miz-1
• induce expression of p15INK4B and weakly
p21Cip1 (CDK inhibitors that shut down
CDK4/6 and CDK2)
• Myc-Miz-1 repress expression of these
CDK inhibitors
• this action can be preemptively blocked
by TGF-β by dispatching Smad 3 to form
complex with E2F 4 or 5 plus p107
(cousin to pRb) > represses myc gene
Cancer cell that have
escaped growth-inhibitory
influence of TGF-β often
have pRb eliminated from
this regulatory circuitry
Rb also controls cell differentiation
• The control of cell cycle differentiation is
coupled to the regulation of cell cycle
progression
• Hypophosphorylated Rb is needed to halt
proliferation of cells and facilitate differentiation
• Dephosphorylation of Rb and blockage of cell
cycle is a "differentiation decision"
• Myoblasts only differentiate after proliferation
stops (e.g. removal of growth factors)
• bHLH transcription factor MyoD forms
heterodimers with E12 and E47 to orchestrate
myocyte differentiation
• Id2 binds to MyoD and prevents differentiation
• Expression of Id2 can be induced by Myc
• In normal cells Id2 is sequestered by pRb
• Overexpression of Id2 in neuroblastomas
81
Perturbation of the R point transition in tumors
favor advance
block advance
The discovery of p53
•
•
•
SV40 transformed tumor cells stained in the nucleus for SV40 large T antigen
Immunoprecipitation of 94 kD protein only from virus infected 3T3 cells, also
a 53 to 54 kD protein was detected in transformed 3T3 cells and both F9 cells
LTA tightly bound to novel protein, now called p53, of cellular origin
Wildtype p53 with ras: no tumor
With mutated p53: tumor
- few tumors with
complete deletion
- robust tumor with point
mutation
3T3 Balb/c mouse fibroblasts
F9 mouse embryonal
carcinoma cells
T = SV40 LTA N = negative
Mutant versions of p53 interfere
with normal p53 function
Tumor-bearing Mice
p53 +/+
p53 +/-
1% at 18 months
2% at 9 months
Disruption of embryonic development
p53 -/-
75% at 6 months
No effect on embryonic development
• p53 does not operate to transduce proliferative and anti-proliferative
signals that continuously impinge on cells and regulate cell proliferation
• p53 seems to specialize in preventing appearance of abnormal cells
82
p53 TUMOR
SUPPRESSOR FUNCTION
Interaction between the p53 protein
and the DNA molecule is required
for tumor suppressor function
Amino acid residues in the p53
protein that contact the DNA
molecule are frequently mutated
Mechanisms of p53 dominantnegative mutations
• p53 normally functions as a homotetrameric transcription factor
• in cells bearing a mutant p53 allele that encodes an altered protein the
structurally altered protein may retain its ability to form tetramers, but
loose its ability to exert normal p53 function
• a single mutant protein subunit may compromise tetramer function
15/16 tetramers with
altered function
Nature of p53 mutations
•
•
•
•
point mutations are most common in p53 > dominant negative effect
striking contrast to mutations in other tumor suppressor genes such as APC
or “caretaker” genes involved in maintenance of the genome (ATM, BRCA)
frame-shift mutations or nonsense codons in the majority of cases
disrupt protein structure and create truncated, often defective proteins
83
Frequency of mutant p53 alleles in
human tumor genomes
p53 responds to various stressful signals
Induction following
DNA damage
p53 mediated apoptosis
• p53 uses multiple signaling
pathways to activate apoptotic
program
• induces expression of genes
encoding:
• Fas receptor
• Insulin like growth factor (IGF)
F-binding protein-3 (IGFBP-3)
• Bax
• display of Fas receptor sensitizes
cell to FasL induced apoptosis
• release of IGFBP-3 from cell causes
binding of pro-survival IGF1 and 2
• Bcl-2 related Bax is pro-apoptotic
and causes release of cytochrome c
p53
84
Control of p53 levels by Mdm2 (mouse double minutes)
• following signaling p53
tetramer binds to the promoters
of target genes including mdm2
• increase in Mdm2 protein
• Mdm2 binds to p53 subunits
• blocks functional ability of p53
• triggers ubiquitilation and after
transport to cytoplasm
degradation in proteasomes
• negative feedback loop
• phosphorylation of p53 blocks
ability of Mdm2 to bind to p53
• by kinases ATM, Chk1 and 2
which become activated by
DNA damage
Consequences of p53 mutations
• Li Fraumeni syndrome (familial cancer)
• susceptibility to a wide variety of
cancers due to mutations in p53
• green: breast cancer red: sarcoma
• blue: lung cancer brown: Wilms tumor
• yellow: glioblastoma purple: leukemia
• orange: pancreatic carcinoma
Cellular
response to
irradition
85
EVADING APOPTOSIS
Differentiation
Stimulated
Inhibited
Proliferation
Cell
Population
Apoptosis
Inhibited
Stimulated
Rates of cell proliferation and cell death in a homeostatic tissue
are roughly equal. Perturbations in this homeostasis (increased
proliferation or decreased death) can result in excessive
tissue growth and ultimately may evolve into a tumor
Tumorigenic Stress
Both extracellular and intracellular
stresses can induce tumorigenic
changes in normal cells. Cells then
attempt to avoid transformation by
undergoing either cell-cycle arrest
(time for the repair of DNA damage)
or cell death.
Several pathways, which involve
some of the same signaling
mediators, exist to prevent
transformation, including those
that lead to apoptosis, mitotic
catastrophe, autophagy and
necrosis.
Anti-apoptotic strategies
• decrease of pro-apoptotic protein
function (labeled blue)
• increase of anit-apoptotic protein
function (labeled red)
• commonly employed mechanisms
by cancer cells to evade apoptosis:
- inactivation of the p53 pathway
- inactivation of Rb function
- shutting down ARF
- deregulation of bcl-2 like proteins
- hyperactivation of the PI3K >
Akt/PKB pathway
- inactivation of PTEN
- activation of NF-κB expression
- expression of FLIP protein
86
Cellular failsafe mechanisms
• aberrantly activated oncogenes induce
failsafe mechanisms in the cell to
prevent unregulated proliferation
• cells exit cell cycle via CDK4 inhibitor
INK4A and Mdm2 inhibitor ARF
• activated Ras or Myc induce p53 and
Rb anti-proliferative activities
• oncogenic RAS leads to upregulation
of INK4A, blocks cyclin-D/CDK4-mediated
hyperphosphorylation of RB, provokes
cellular senescence
• activated MYC induces ARF, blocks
p53 inhibitor Mdm2, activates p53
• tumorigenesis relies on the cancellation
of these failsafe mechanisms
Control of apoptosis by ARF
• ARF (p14ARF) can
associate with Mdm2
• drags Mdm2 into
nucleolus > can’t
attack p53 there >
×ARF = ×p53
• numerous oncogenic
signals (e.g. Myc, Ras)
favor apoptosis and
induce E2F activity
• × ARF expression
• inactivation of one
allele of ARF > rapid
tumor development in
mice with Eµ-myc
transgene
Actions of the anti-apoptotic Bcl-2 gene
• clones of myc and bcl-2 were placed
under control of promoter IgG >
expression in B cell lineage
• induced in germ lines of mice
• mice carrying various transgene
• no mortality in bcl-2 transgenes, greatly
increased mortality in myc transgenes
• acceleration of
lymphomagensis
in mice with myc
and bcl-2 transgene
• bcl-2 prolongs the
life of lymphocytes
that would otherwise undergo
apoptosis
87
Actions of the anti-apoptotic Bcl-2 gene
• chromosomal breakpoint in human
follicular B-cell lymphomas
• translocation generates a fusion
between Ig gene on chromosome
14 and the bcl-2 gene on
chromosome 18
• high levels of expression of bcl-2 mRNA in mature B-cells
• increased Bcl-2 protein levels
impedes apoptosis in B-cells,
normally a high turnover rate
• end result: follicular lymphoma
• relatively indolent cancer
(small resting B-cells)
• often progresses to malignant
high grade lymphoma
Exploitation of apoptosis by cancer
therapy treatments
• Eµ-Myc mouse tumors treated with DNA damaging agents
• trigger a p53-mediated apoptotic response, tumor burden is lowered
• apoptotic response is prevented by Bcl-2 overexpression
Rapamycin and the mTOR circuit
• mammalian target of rapamycin
• signals: nutrients, oxygen, ATP,
mitogenic signals
• responses: cell proliferation,
protein synthesis, angiogenesis,
anti-apoptotic, ribosome
biogenesis, cell motility
• exists in alternative complexes:
• Rictor > activity of Akt/PKB
• Raptor > activates protein
synthesis
• rapamycin blocks mTOR-Raptor
• also shuts down mTOR-Rictor
• primary effect is inhibition of
Akt/PKB signaling (carcinomas
with loss of PTEN)
88
Mitotic catastrophe as a failsafe mechanism
• type of cell death caused by aberrant mitosis: characterized by
multinucleate giant cells that contain uncondensed chromosomes
• integrity of M-phase progression is monitored by:
• detection of DNA damage (G2/M arrest)
• detection of mitotic-spindle malformations
• detection of incorrect positioning of the spindle
• defects in genes required for MC can contribute to tumorigenesis
Mitotic catastrophe as a failsafe mechanism
• numerous kinases involved in mitotic regulation: polo-like kinase 1
(PLK1), aurora kinase family (aurora-A and -B), regulator of spindle
checkpoint called BUB-related kinase (BUBR)
• response to genotoxic stress: ataxia teleangiectasia mutated (ATM) and
ATM- and Rad3-related (ATR) are activated and activate the checkpoint
kinases CHK1 and 2 > phosphorylate and inactivate CDC25 >
translocated into the cytosol > blocks CDK1 activation > G2 arrest
Consequences of DNA damage
• results in immediate arrest in the interphase of the cell cycle or can
cause a transient entry into the prophase (reversible chromatin
condensation) with a prolonged 'antephase‘
• failure to activate the checkpoints, causes entry into mitosis and death
during the metaphase
• in case of mitotic slippage and consequent mitotic failure, generation of
tetraploid cells that undergo apoptosis or generate aneuploid cells
89
Autophagy and beclin 1
• adaptation to nutrient limitation by catabolizing intracellular components
to ensure survival, also targeted elimination of proteins and organelles
• can lead to cell death under prolonged nutrient starvation
• Beclin-1 is a key regulator of the autophagy process
• activated by nutrient limitation or damaged or excess organelles
• wortmannin and 3-methyladenine inhibit beclin-1 > inhibit autophagy
• 40-75% of breast or ovarian carcinomas have deletions of beclin-1 gene
Autophagosome
Autolysosome
2) Nucleation/ 3) Membrane
Assembly
elongation
4) Fusion
5) Degradation
Senescence and Immortality
Replicative senescence
a non-growing state of cells in
which they exhibit distinctive
cell phenotypes and remain
viable for extended periods of
time, but are unable to
proliferate again
loss of proliferative
capacity with age
Immortality
trait of a cell or population of
cells to proliferate indefinitely
Telomeres
• telomeres are TTAGGG repetitive sequences that cap the
end of eukaryotic chromosomes
• protect the ends of chromosomes from degradation and from
end-to-end fusion with other chromosomes
• a core of telomere binding proteins termed the sheltering
complex, serve to protect telomeric ends
• critical shortening or uncapping of telomere binding proteins
results in telomere dysfunction
• dysfunctional telomeres activate a DNA damage response
• in cells with functional p53 pathway, this initiates cell crisis
and apoptotic programs to inhibit tumorigenesis
• in cells with mutant p53, dysfunctional telomeres promote
genomic instability and progression to cancer
90
Replicative lifespan
• normal cells: mortal, no telomerase activity,
telomere shortening with divisions
• cancer cells: immortal, express telomerase,
stable telomere lengths
• normal cells have limited replicative lifespan,
enter irreversible growth arrest, replicative
senescence (M1) after extended passage
• introduction of viral oncoproteins (SV40)
before M1 allows continued proliferation with
further shortening of telomeres > second
proliferative barrier, crisis (M2)
• crisis: state arising when cells lose
telomeres of adequate length, resulting in
end-to-end fusion of chromosomes,
karyotypic chaos, and widespread apoptosis
• activation of telomerase through introduction
of the catalytic subunit of telomerase, TERT,
allows cells to avoid replicative senescence
Breakage-Fusion-Bridge (BFB) cycles
• occurs when telomeres are too short to protect
chromosomal DNA (in G2 phase, telomeres
were eroded in duplication in S phase)
• during normal mitosis 2 sister chromatides are
pulled apart to opposite centrosomes
• centromeres in fused chromosome will also be
pulled apart
• dicentric chromosomes
are unable to separate
• create bridge between
poles of mitotic spindle
• break at weak point
• new fusions in next
cell cycle
• repeat of earlier events
• BFB cycles create
karyotypic chaos
Telomere structure
• TTAGGG repetitive sequences that
terminate in a 3’ single-stranded
overhang (ss)
• ss overhang can invade the double
stranded region of the telomere to
form a protective telomere loop at
the invasion site (t loop)
• telomeric DNA is complexed by the sixprotein-sheltering complex of telomericrepeat binding factors 1 and 2 (TRF1 and
2), RAP1, TRF1-interacting nuclear factor
(TIN2), TPP1, POT1
• TPP1-POT1 heterodimer regulates access
of telomerase to the telomeric substrate
• telomeres transiently interact with a host
of other factors, many of which are
involved in DNA damage response
G-rich strand
91
End replication problem
• during DNA replication the parental DNA double-helix is unwound by helicase
• new lagging strand synthesis by short RNA primers that lay down intervals
• new leading strand synthesis can proceed continuously (5’ to 3’ direction)
• also initiated by an RNA primer that sits on 3’ end of parental strand
• loss of primer binding site, “under-replication” of parental strand
Telomere dysfunction
• progressive shortening of telomeres
initiates DNA damage response
• activation of ataxia telangiectasia mutated
(ATM) and ataxia-telangiectasia and -Rad3related (ATR) and kinases CHK1 and 2
• uncapping of TRF2 engages ATM
• uncapping of POT1 engages ATR
• phosphorylation of p53 > regulation of
senescence and apoptosis
• transcription of p21 induces a senescentlike growth arrest, cannot be reversed by
physiological mitogens, but is reversible
upon inactivation of p53
• oncogenes and other types of stress
induce p16, which activates pRB
• pRB-mediated senescence arrest cannot be
reversed by inactivating p53, pRB, or both
Telomerase
• the telomerase holoenzyme is
composed of at least 2 essential
subunits
• hTERT (human telomerase reverse
transcriptase) catalytic subunit and
the hTR RNA subunit (telomeraseassociated RNA molecule)
• the holoenzyme attaches to the 3’
end of the G-rich strand overhang
• reverse transcription of sequences
present in hTR subunit
• extends the G-rich strand by 6
nucleotides
• process is repeated hundreds of
times
92
LIMITLESS REPLICATIVE POTENTIAL
• ss overhang degraded in cells
entering replicative senescence
• blunted DNA emits damage signal
• activation of p53
• p16INK4A which is activated by
physiologic stress
• together they induce senescent
growth state
• telemorase expression, catalytic
subunit hTERT) can prevent cells
from entering into replicative
senescence by reversing loss of
G-rich overhang
• cells in culture with ectopically
expressed telomerase continue to
grow, proliferate indefinetely
LIMITLESS REPLICATIVE POTENTIAL
• some cells maintain telomeric DNA
without actions of telomerase
• alternative lengthening of telomeres
(ALT) used by some cancer cells
• DNA polymerases use more than
one template during replication
• one telomere extends its overhang,
which displaces a strand of same
polarity in the telomere of another
chromosome (3’ end anneals)
• conventional polymerases extend
strand using complementary strand
as template
• the recently elongated strand
disengages and can be converted
into double strand
• process is repeated multiple times
copy choice mechanism
BFB cycle promotes carcinoma formation
• inactivation of p53 during early stages of
tumor progression (favors escape from
apoptosis due to oncogene activation etc.)
• as tumor progression proceeds, the
telomeric DNA of pre-malignant cells will
erode beyond chromosomal protection
• BFB cycles will ensue
• leads to increased chromosomal
rearrangement and amplification and
deletion of chromosomal segments close
to breakpoints
• cells not eliminated by p53 (see above)
• variants will emerge that can regenerate
telomeres and thereby stabilize their
karyotype, cells that can grow rapidly
• activation of ALT mechanism, derepression of hTERT expression
93
TISSUE INVASION
AND METASTASIS
Cancers grow by progressive
infiltration, invasion, destruction, and
penetration of the surrounding tissue
SPREAD OF NEOPLASMS
Local invasion
Implantation
Hematogenous
Lymphatics
Risk of metastasis in
correlation to primary
breast carcinoma size
SPREAD OF NEOPLASMS
Direct invasion of normal lung by lung SCC (right),
and tumor spread through bronchioles (left)
94
SPREAD OF NEOPLASMS
Infiltration and dissection along tissue planes
Requires a lack of cohesiveness between cells
Enzymatic degradation of extracellular matrix
Extension of pseudopodia between cells
Mesothelioma on the pleural surface of the lung
SPREAD OF NEOPLASMS
Lymphatic spread and lymph node involvement by metastatic
tumors will reflect the natural lymphatic drainage of the tissue
site of the primary tumor.
Mammary carcinoma spreading through a lymph node (left), and
mammary carcinoma in a dilated lymphatic vessel of the lung (right)
SPREAD OF NEOPLASMS
Hematogenous spread occurs principally through the
venous circulation. Consequently, the liver and lungs
are the most frequently involved secondary sites.
Invasive mammary carcinoma metastatic to liver.
95
ORGAN PREFERENCES
FOR METASTASES
• Hemodynamic and Anatomic factors
• Filtering organs often sites of metastasis:
• Extensive capillary network of the lung and liver
• Regional lymph nodes
• Sites of endothelial damage:
• Interaction of neoplastic cells with platelets and fibrin
• Tissues with large volume of blood flow:
• Brain and Kidney
ORGAN PREFERENCES
FOR METASTASES
• Intrinsic factors of neoplastic cells
• Neoplastic cells need hospitable environment to
colonize: “seed and soil theory” (contralateral mets?)
• Neoplastic cells may possess tissue-specific cell
membrane proteins which interact with endothelial
cell proteins to facilitate metastasis: vascular ZIP code
• Metastatic sites may be pre-programmed and not
random: “The coconut theory”
ORGAN PREFERENCES FOR METASTASES
96
Metastases to bone
• requires subversion of
osteoclasts and osteoblasts
• osteolytic or osteoblastic lesions
• receptor activator of NF-κB ligand
(RANKL) is important mediator of
osteoblastic differentiation
• RANK binding on osteoclasts
precursors to RANKL on
osteoblasts results in
differentiation to functional
osteoclasts: osteolysis
• osteoprotegerin (OPG) can
prevent RANKL binding
• OPG – RANKL balance determines
the net rate of bone growth/loss
Osteolytic bone metastases
• release by tumor cells of PTHrP >
acts on osteoblasts > increase
RANKL release, blocks OPG >
maturation of osteoclasts > bone
lysis > exposure of bone ECM >
release of TGF-β1, Ca2+, IGF-1,
PDGF, FGFs, BMPs > tumor cell
survival and proliferation
• TGF-β1 initiates positive feedback
loop > release of more PTHrP
• “vicious cycle” of osteolytic mets
• breast cancer, anal sac carcinoma
• Prostate cancer causes osteoblastic
metastases through release of
endothelin 1 growth factor
Metastatic tropism and gene expression
IL11 (interleukin11), OPN (ostopontin),
CTGF (connective tissue growth factor),
CXCR4 (chemokine receptor 4), MMP1
(matrix metallo proteinase 1)
Original tumor
Metastatic subclone
• 33 single cells from
mammary carcinoma
clonally expanded
• analyzed mRNA pattern
• visualization of mets with
luciferase gene
• 5 genes analyzed since
they are overexpressed
in bone metastases and
promote osteolysis
• bone metastases: all 5
genes are high (clone 2),
lung metastases: all 5
genes are low (clone 3),
no metastases: no gene
expressed (clone 26)
GAPDH (glyceraldehyde 3 phosphatase
dehydrogenase)
97
SPREAD OF NEOPLASMS
The invasion-metastatic
cascade
Most malignant cells released from
tumors die in circulation
Detachment of tumor cells
from each other
- E-cadherins mediate adhesions
- linked to cytoskeleton by catenins
Attachment to matrix
components
- receptor-mediated
- to laminin and fibronectin
- express higher amount of integrins
- produce integrins that are not
present in the normal tissue
Degradation of ECM
- chemical alteration of connective
tissue matrix
- 3 classes of proteases: serine,
cysteine, and matrix
metalloproteinases (MMPs)
- MMPs promote angiogenesis,
tumor growth, tumor cell motility
Patterns of local invasion
• individual cells leave primary
tumor one by one (“Indian file”)
through channels in stroma
• phalanx (“well organized cohort”)
of cells invades nearby stroma,
more typical behavior
E-cadherin
metastatic melanoma cells
collagen
β1 integrins
98
Steps leading to extravasation
cancer cells are physically trapped in capillaries > attach to platelets >
form microthrombus > degranulate, release growth factors, proteases etc.
> push aside endothelial cell on one side of wall (contact to basement
membrane) > microthrombus is dissolved by proteases > proliferation of
cancer cells in lumen > break through basement membrane
Tumor progression
Darwinian evolution and
clonal expansion of
advantages genotypes
and thus phenotypes
Selection, outgrowth
- more growth
autonomous
- ignore death and
senescence signals
- escape immune
surveillance
- trigger angiogenesis
- invasion, metastasis
Nonviable (antigenic, apoptotic, etc.)
The evolution of colonizing ability
Initially formed genetically heterogenous
primary tumor cell population seeds
equally heterogeneous micrometastases
Surgically removed primary tumor leaves
behind minimal residual disease
Micrometastasis aquires ability to
colonize, i. e. grow into macrometastasis
New source for micrometastases that are
genetically very similar to another
Grow fast new macrometastases since
the have ability to colonize
99
Epithelial-mesenchymal transition
• shedding of epithelial
characteristics to acquire
motility and invasiveness
• in normal epithelium Ecadherin is tethered to
actin cytoskeleton via αand β-catenin
• loss of E-cadherin
liberates β-catenin which
migrates to the nucleus
• associated with Tcf/Lef
transcription factors and
key intermediary in Wnt
pathway
• initiates EMT program
β-catenin and the biology of colonic crypts
•
•
•
•
•
Stem cells in crypts: high levels of
β-catenin, signaled by stromal Wnt
β-catenin associates with Tcf/Lef
transcription factors > increased
proliferation > cells migrate to
lumen > decrease in Wnt and
increase in Apc > increased
degradation of β-catenin > cessation
of proliferation and apoptosis
Apc (adenomatosis polyposis gene)
negatively controls β-catenin levels
Apc brings together glycogen
synthase kinase 3β and β-catenin >
phosphorylation of β-catenin >
degradation by ubiquitin-proteasome
pathway
Apc inactivation leads to high levels
of β-catenin
Cadherin shift and invasiveness
• shift from E- to N-cadherin in melanomas enables migratory behavior
• shift facilitates invasion into stroma since melanocytes can dissociate
themselves from neighboring keratinocytes without E-cadherin
• N-cadherin allows homotypic interactions with various cell types
100
Control of EMT by TGF-β
• Ras transformed mammary tumor cells express E-cadherin, but when
grown in presence of TGF-β they undergo EMT and express vimentin
• Ras downstream effectors Raf and PI3K induce TGF-β secretion
• tumor cells express αVβ6 integrin while TGF-β is expressed by stroma
• αVβ6 activates latent TGF-β in stromal cells > positive feedback loop
Role of NF-κB in EMT
• Ras transformed mammary tumor cells express E-cadherin, but TGF-β
suppresses E-cadherin and induces vimentin expression
• NF-κB signaling is suppressed > TGF-β fails to suppress E-cadherin
• NF-κB is necessary for EMT and constitutive activation of NF-κB will
induce EMT in cancer cells
Macrophages stimulate invasive behavior
• breast cancer cells recruit large
numbers of tumor associated
macrophages (TAM)
• TAMs are found in close
proximity to microvessels and
are the major source of EGF
• EGF stimulates cancer cells to
release colony stimulating
factor (CSF-1)
• as a consequence CSF-1
stimulates EGF production:
positive feedback loop
• TAM derived TNF-α further
contributes to EMT
• TAMs also release MMPs
101
Macrophage contribution to tumorigenesis
• carcinoma cells recruit circulating
monocytes into tumor where they
differentiate into macrophages (TAM)
• recruited by chemotactic factors:
• monocyte chemotactic protein (MCP-1)
• colony stimulating factor (CSF-1)
• platelet derived growth factor (PDGF)
• TAMs secret EGF > cancer proliferation
• hypoxic areas attract TAMs > secret
VGEF > reduces hypoxia by bringing in
endothelial cells > angiogenesis
• TAMs secret MMP-9 (gelatinase B) and
degrade ECM and cleave other proteins
• Cleave insulin like growth factor
binding proteins (IGFBP) > liberates
IGF > survival signals to cancer cells
Invasive behavior induced by HGF
• hepatocyte growth factor
(scatter factor: SF) is
produced by a wide variety
of stromal cells
• Met is the cognate receptor
expressed on epithelial cells
• epithelial cells stimulated
with HGF become motile
and scatter through growth
medium
• in collagen gels HGF
stimulated cells invade into
surrounding gel
Signals that trigger EMT
• TNF-α, EGF, HGF, FGF
induce Ras pathway that
stimulates TGF-β and
blocks apoptotic effect
of TGF-β through PI3K
• NF-κB, TGF-β and β–
catenin induce EMT in
cancer cells: motility and
invasiveness
• not only genotype
dictates cancer cell
phenotype, but also
heterotypic interactions
(depend on normal cells)
and microenvironment
102
Embryonic EMT signaling
• striking similarities in EMT
signaling between early
embryogenesis and
tumorigenesis
• further support for the
notion that the EMT
program expressed by
invasive carcinoma cells
represents a reactivation of
latent cell-biological
programs, many of which
are active in early
mammalian embryonic
development
Extracellular proteases in invasiveness
• proteases are key effectors of EMT
• mainly matrix metallo-proteinases (MMPs)
• produced by macrophages, mast cells,
fibroblasts and other stromal cells
• some cancer cells may have induced
synthesis of MMPs, mainly MMP-2 and 9
• MMPs cleave collagens, laminin, tenascin,
fibronectin and proteoglycans
• synthesized as inactive zymogens and
activated by proteinase cleavage
• 187 MPs: only 28 are secreted MMPs,
6 are membrane-anchored (MT1-MMP)
• MT1-MMP can only cleave proteins in
close proximity to cell
• can activate pro-enzymes like pro-MMP2
• activity can be confined: podosomes
Urokinase plasminogen activator (uPA)
• inactive pro-enzyme of uPA is
released from stromal cells
• binds to its cognate receptor, uPAR,
which is displayed on epithelial cells
• binding converts uPA into catalytic
active protease that can convert
plasminogen into active plasmin
• plasmin functions as protease to
cleave pro-enzyme forms of MMPs
and latent form of TGF-β
• active forms degrade ECM
• there is also evidence that uPA can
directly convert pro-MMPs
103
MMPs in tumorigenesis
• proteases are mainly effectors of EMT, but can drive progression of
cells through all stages of multi-steps tumorigenesis
• in particular MMP-3 (stromelysin-1)
• overexpression of MMP-3 in mammary glands of transgenic mice will
lead to hyperplasia and eventually progress to malignancy
Locomotion on solid surfaces
• locomotion of cultured cells
depends on coordination of a
complex series of changes in
cytoskeleton and breaking of focal
contacts with growth substrate
• cell organizes actin fibers to
extend lamellipodia on edge
• filopodia protrude from these
• cell surface proteases degrade
proteins that stand “in the way”
• integrins establish new points of
contacts at lamellipodia edge
• actin and myosin II stress fibers
contract cell on trailing edge to
break focal contacts
The circuitry mediating cell motility
• motogenic growth factors EGF,
HGF and PDGF stimulate circuitry
• Rho family of small GTPases ( i.e.
Rho, Rac, Cdc42) has central role in
controlling actin cytoskeleton and
focal adhesions
• stimulation of Ras effector PI3K
allows guanine nucleotide exchange
factors (GEF) to attach to membrane
and to activate Rho GTPases
• Tiam1 (T cell lymphoma and
metastasis gene) is stimulated by
Ras and PIP3
• Tiam1 GEF polymerizes actin on
leading edge of migrating cells
104
Metastasis suppressor genes
• relatively little know, early stage of research
• many genes encode growth factors, growth factor receptors and
signal-transducing proteins that when introduced in epithelial cells
encourage EMT, acquisition of motility and invasiveness
• deregulated versions of these genes may drive metastasis and invasion
• mechanisms of E-cadherin or TIMP seem obvious, others are unknown
SUSTAINED ANGIOGENESIS
• oxygen, amount of nutrients, ability to
shed metabolic waste products require
close proximity to vasculature
• necrosis is the primary result of hypoxia
in poorly vascularized areas
• oxygen pressure drops to zero at a
distance of more than 0.2 mm
• tumor can not only depend on genetic
blue print for growth, but has to rely on
heterotopic interactions when designing
the layout of its own vasculature supply
• endothelial cells, pericytes and smooth
muscle cells all play a critical role in
tumor angiogenesis: VEGF, TGF-β, basic
fibroblast growth factor (bFGF), IL-8,
angiopoietin, angiogenin, PDGF
Angio- and lymphangiogenesis
• angiogenesis (the formation of new blood vessels from existing ones)
is an important process in the growth of malignant tumors
• an association between VEGF and angiogenesis, malignancy, and
metastasis has been established
• many pro- and anti-angiogenic cellular factors regulate angiogenesis
• major steps of endothelial cells in angiogenesis:
- breaking through of basal lamina that
envelops existing blood vessels
- migration towards source signal
- proliferation - formation of tubes
• blood and lymphatic network derive from
common precursors in embryo
• VGEF-A and -B stimulate vascular growth
• VGEF-C and -D stimulate lymphatics
• VGEFs and VGEF-Rs are homologous
• no support by mural cells in lymphatics
105
Tripping the angiogenic switch
• “angiogenic switch”: moment at
which a tumor begins to overexpress
pro-angiogenic factors such as VEGF
• malignant tumors need to attract
blood vessels to grow beyond 0.2 mm
• transgenic Rip-Tag mouse model:
- normal pancreatic islets have small
number of capillaries for support
- 50% of islets: hyperplasia (-0.2 mm)
- remain in dynamic no growth state
- islet cells express VGEF which is
sequestered by ECM
- 10% islets become angiogenic
- recruit mast cells and macrophages
- both release MMP-9 > activate VGEF
- 3% malignant transformation
Angiogenic switch initiates a complex process
• angiogenesis involves stroma prior
to breakdown of basement membrane
• PIN (carcinoma in-situ) recruits
accumulation of stromal cells
through intact basement membrane
• tumor invasiveness and angiogenesis
are tightly coupled
• microvessel density is commonly
associated with malignancy
• tumor needs to recruit endothelial
precursor cells (EPC) from bone
marrow, stimulated by VGEF and
stroma-derived-factor-1 (SDF-1) that is
released from myofibroblasts
• EPC reach tumor through blood
circulation and differentiate
Prostate intraepithelial
neoplasia (PIN)
Angiogenesis inhibitors
• angiogenesis is normally suppressed
by physiologic inhibitors
• suppression of hypoxia-inducedfactor-1 (HIF-1) central in healing
• ECM components block angiogenesis:
• thrombospondin-1 (Tsp-1) binds to
CD36 on endothelial cells and halts
proliferation
• Tsp-1 also releases Fas ligand from
endothelial cells > apoptosis
• Fas receptor only on proliferating cells
• Tsp-1 is induced by p53 and can be
shut down by Ras oncogene
• endostatin, endorepellin, troponin 1,
tumstatin, arresten, TIMPs etc.
• non-matrix components: IFN-α, IL-1, etc.
106
Balancing the angiogenic switch
• diagram of major physiologic
regulators that promote or inhibit
angiogenesis
• in reality not a binary decision, but
switch has many gradations of
development during tumorigenesis
• highly complex systems are more
vulnerable to disruption
• at least a dozen factors involved in
regulating vascular morphogenesis
• complexity offers multiple targets
for intervention
• much easier to target angiogenesis
since normal cells are targeted and
not tumor cells that commonly
become therapy resistant
Therapeutic considerations
• anti-VGEF-R drug effective on blocking
angiogenic switch in hyperplastic islets
• anti-PDGF-R-drug more effective late stage
• VGEF-R only important in early stages of
angiogenesis, but PDGF-R vital in recruiting
pericytes to growing capillaries, protects
them against anti-angiogenic signals
anti- VGEF-R and PDGF-R
combination therapy
Therapeutic considerations
Heterotopic interactions
as target for therapy
107
THE ROLE OF THE STROMA
Cross talk between the ECM and tumor cells
cleavage of matrix components (type IV collagen),
degradation of laminin by MMP-2 release angiogenic factors
and proteolytic fragments that favor cancer cell motility
ECM stores growth factors in inactive forms
PDGF, TGFβ, and b-FGF
affect the growth of tumor cells in a paracrine manner
Stromal cells transmit oncogenic signals
Stroma can drive genetic changes that promote
carcinogenesis
CARCINOGENESIS IS A MULTISTAGE
PROCESS
Initiation Promotion Conversion
Progression
Defects in Cellular Differentiation
Defects in Growth Control
Resistance to Cytotoxicity
Insult
Genetic
Change
Normal
Cell
Selective
Clonal
Expansion
Genetic
Change
Genetic
Change
Initiated Preneoplastic Malignant
Cell
Lesion
Tumor
Genetic
Change
Clinical
Cancer
Advanced
Clinical
Cancer
Activation of Proto-oncogenes
Inactivation of
Metastasis
Inactivation of Tumor Suppressor Genes
Suppressor Genes
Colorectal cancer
• a series ranging from single crypt lesions (aberrant crypt foci) through
small benign tumors (adenomatous polyps) to malignant cancers
• inherited factor : HNPCC (hereditary nonpolyposis colorectal cancer)
and FAP( familial adenomatous polyposis)
• activation of RAS oncogenes (50 % of colorectal cancers, c-Kis-RAS,
N-RAS) and inactivation of TSGs on ch 5q (p53 gene), 17p (DCC,
SAMD4/DPC4, SMAD2)
• mutation of APC-tumor suppressor gene on ch 5q, increased
β-catenin/Tcf-mediated transcription
• several inherited predispositions can result from inheritance of a
single defective gene
– caretakers (DNA MMR gene in HNPCC), gatekeepers (APC),
• definition of a model for colorectal tumor development
– mutations of APC→K-RAS→18q suppressor and p53
• accumulation of genetic changes is facilitated by a chromosomal
instability
108
Inherited predisposition
1. Presence of polyposis (FAP)
• develop hundreds to thousands of adenomatous polyps during their
lifetime
• colorectal cancer develop at median age of about 40
• increased risk for thyroid, small intestine, stomach, and brain
• GS (Gardner syndrome), CHRPE, Turcot syndrom are variants of FAP
• germ-line mutations of the APC gene
2. Absence of polyposis (HNPCC)
• develop at median age of about 42 years
• defect in DNA MMR genes
• at increased risk for uterus, ovary, brain and other cancers
Other genetic Factors and environmental factors(diets)
• associated with increased risk for colorectal cancer
The APC gene
• expression increases as cells migrate to top of crypt
• β-catenin, γ-catenin, GSK-3β, AXIN family proteins, EB-1
and hDLG bind to the C-terminus of APC
→ all APC mutations result in loss of the C-terminus of
APC → no interaction
• mutation of APC : prevent its inhibition of β-catenin
• dominant activating β-catenin mutations that render the
protein insensitive to APC/GSK-3β-mediated degradation
• increased β-catenin/Tcf mediated transcription results in
expression of genes that promote cell growth or inhibit
cell death (ex. c-MYC)
• APC binds to tubulin and stabilize the microtubule,
possibly involving the chromosomal stability
THE PATHOGENESIS OF HUMAN
COLORECTAL CANCER
Normal
Colonic
Mucosa
Hyperplasia
Intermediate
Adenoma
Late
Adenoma
Carcinoma
Metastasis
109
THE PATHOGENESIS OF HUMAN
COLORECTAL CANCER
CANCER STEM CELL THEORY
• Stem cells as a source of neoplasms
• Neoplasms don’t usually develop from completely
differentiated cells that undergo de-differentiation
• Cells within a tumor that have the capacity to
initiate and sustain the tumor
• These cells maintain their self-renewing capacity,
have the ability to invade surrounding tissue and
evade apoptosis
“ONCOGENY AS PARTIALLY BLOCKED ONTOGENY”
CANCER STEM CELL THEORY
Normal stem cells
Rare cells within organs with the ability to selfrenew and give rise to all types of cells within
the organ to drive organogenesis
Cancer stem cells
Rare cells within tumors with the ability to selfrenew and give rise to the phenotypically
diverse tumor cell population to drive
tumorigenesis
110
Properties shared by normal stem cells
and cancer stem cells
Assymetric Division:
• Self renewal
- Tissue-specific normal stem cells must self-renew throughout
the lifetime of the animal to maintain specific organs
- Cancer stem cells undergo self-renewal to maintain tumor
growth
• Differentiation into phenotypically diverse mature cell types
- Give rise to a heterogeneous population of cells that compose
the organ or the tumor but lack the ability for unlimited
proliferation (hierarchical arrangement of cells)
• Regulated by similar pathways
- Pathways that regulate self-renewal in normal stem cells are
dys-regulated in cancer stem cells
Two general models for cancer heterogeneity
1.
All cancer cells are potential cancer stem
cells but have a low probability of
proliferation in clonogenic assays
2.
Only a small definable subset of cancer
cells are cancer stem cells that have the
ability to proliferate indefinitely.
Two general models for cancer heterogeneity
Self renewal and differentiation are random.
All cells have equal but low probability of
extensive proliferation. Only cells with self
renewal capacity can sustain tumor growth.
Distinct classes of cells exist within a
tumor. Only a small definable subset,
the cancer stem cells can initiate
tumor growth.
111
Pathways involved in self-renewal that are
deregulated in cancer cells
• Wnt, Shh, and Notch
pathways contribute
to the self-renewal of
stem cells in a variety
of organs
• dysregulating these
pathways contributes
to oncogenesis
• mutations of these
pathways have been
associated with a
number of tumors:
• colon carcinoma
(Wnt), basal cell
carcinoma (Shh),
leukemia (Notch)
Development of hematopoietic stem cells
• subdivided into long-term
and short-term selfrenewing HSCs and
multipotent progenitors
• give rise to CLPs (common
lymphoid progenitors)
and CMPs (common
myeloid progenitors
• CMPs/GMPs (granulocyte
macrophage precursors)
and CLPs can give rise to
all known dendritic cells
• MEP = megakaryocyte
erythrocyte precursor
Stem
Cells
CD34+
CD38-
Multipotent
Progenitors
Oligolineage
Progenitors
CD34CD38+
Mature
Cells
CD20+
CD8+
CD8+
Differentiation
Self Renewal
CD34CD38-
CD4+
CD4+
CD36+
CD35+
The importance of self-renewal in leukemic
initiation and progression
Self-renewal is a key property
of both normal and leukemic
stem cells. Fewer mutagenic
changes are required to
transform stem cells in which
the self-renewal machinery is
already active (a), as compared
with committed progenitors in
which self-renewal must be
activated ectopically (b). In
addition, self-renewing stem
cells are long-lived; thus, there
is an increased chance for
genetic changes to accumulate
in individual stem cells in
comparison with more mature,
short-lived progenitors.
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Hematopoietic
Cancer
Stem Cells
Acute myeloid leukemia
(AML) – CD34+ CD38-
Leukaemic Mouse Models:
chronic myelomonocytic leukaemia (CMML)
MRP8-BCL-2
acute myeloid leukaemia (AML)
MRP8-BCL2Xlpr/lpr
chronic myeloid leukaemia (CML)/Blast MRP8-PML-RARα
acute promyelocytic leukaemia (APML)77 MRP8-BCRablXBCL-2
CANCER STEM CELL THEORY
Clonal Genetic Model of Cancer
Epigenetic Progenitor Model of Cancer
tumor-progenitor genes = TPG
gatekeeper mutation = GKM
tumorsuppressor gene = TSG
oncogene = ONC
Therapeutic implications of Cancer Stem Cells
• Most therapies fail to
consider the difference in
drug sensitivities of cancer
stem cells compared to
their non-tumorigenic
progeny
• Most therapies target
rapidly proliferating nontumorigenic cells and
spare the relatively
quiescent cancer stem
cells
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Chronic inflammation and infection can increase cancer risk
Inherited
Disease
Acquired
Tumor Site Risk
Hemochromatosis
Liver
Hereditary Pancreatitis Pancreas
Ulcerative Colitis
Colon
Crohn’s Disease
Colon
219
53
6
3
“18% of human cancers, i.e.,
1.6 million per year, are related to
infection.”
- B. Stewart and P. Kleihues
World Cancer Report, IARC
Press, p. 57, 2003
Disease
Tumor Site
Viral
Hepatitis B
Hepatitis C
Liver
Liver
Bacterial
Helicobacter Pylori Gastric
PID
Ovary
Parasitic
S. hematobium
S. japonicum
Liver Fluke
Risk
88
30
11
3
Urinary Bladder 2-14
Colon
2-6
Liver
14
Chemical/ Physical/Metabolic
Acid reflux
Esophagus
Pancreatitis
Pancreas
Asbestos
Lung pleural
Obesity
Multiple sites
50-100
17
>10
1.3-6.5
Contributions of free readicals to carcinogenesis
• introduction of DNA Damage
• activation of the protective p53 stress
response pathway
• mutation of cancer-related genes
• activation of the NFκB pathway
• activation of COX2 and the arachidonic acid cascade
• activation of the hypoxic protective state by
stabilization of HIF1α
• promote clonal selection of cells resistant to the toxic
effects of free radicals, e.g., p53 mutant cells
Downstream effects of NF-kB
114
Mechanisms of activation of NF-kB
Free Radicals
Epithelial inflammation and tumor promotion
•
•
•
•
•
inflammatory stimuli, including
chronic infection, result in
recruitment of inflammatory
cells into areas of inflammation
TNF-α released by inflammatory
and endothelial cells
activation of NF-κB pathway
production of more TNF-α, antiapoptotic protein (e.g. Bcl-XL),
mitogenic proteins (e.g. Myc)
also production of COX-2, which
synthesizes multiple
prostaglandines that induce
different cancer phenotypes:
loss of contact inhibition,
increased proliferation,
anchorage-independent growth
Innate Immune
Response
Experimental pathway
12-O-tetradecanoylphorpbol-13-acatate)
Stress
Adaptive Immune
Response
Inflammation
• Cytokines
• Chemokines
• Free Radicals
• Prostaglandins
• Growth Factors
• MMPs
Epithelium
Adaptive Immune
Response
• Cytotoxic T cell
Activity
• Tumor Cell Lysis
• Humoral Immune
Response
• T-Reg Cells
Stroma
• DNA Damage
• Protein Modification
• Proliferation
• miRNA Expression
• DNA and Histone Methylation
• Angiogenesis
CANCER
Landscape
Effects
Metastasis
115
Molecular Based Diagnostic Tests in
Canine Hematologic Malignancies
• Detection of individual mutations
in oncogenes: c-kit, p53
• Detection of chromosomal
translocations, deletions and
duplications: bcr-abl
• Detection of clonality in
lymphomas
Response to Cytotoxic Drugs
p53
Response to Cytotoxic Drugs
Caspase 3
116
bcr-abl Translocations Producing a Fusion Protein
• abl gene (Abelson murine leukemia virus), rapidly
tumorigenic retrovirus, maps to chromosome 9q34
• Fused with sequences clustered at 22q11, called
breakpoint cluster region = bcr (both are multidomain,
multifunctional proteins)
• Identified in 95% of Chronic myelogenous leukemia
(CML)
• Expression of bcr/abl gene driven by bcr gene
promoter, produced transcript represents a chimera
consisting of 5’ portion of bcr gene, and 3’ portion of cabl
• bcr/abl protein expresses enhanced tyrosine kinase
signaling activity compared to the normal ABL protein
(gain of function)
Feline TCRG V-N-J alignment CDR3 region
Moore et al., Vet Immunol Immunopathol 106: 167-178, 2005
PCR
5’ primer
3’ V segment
3’ primer
N region
J segment
CDR3
117
Molecular Clonality - Indications
• Morphological, cytological,
immuno-phenotypic properties
inconclusive
• Lack of architectural effacement in
organized lymphoid tissue - MZL or TZL
• Lympho-histiocytic proliferations in skin
• Lamina proprial or intra-epithelial
lymphocytosis in the small intestine
Molecular clonality - Limitations
• Sensitivity limited with high polyclonal
background
• Miss small clonal populations
• Sensitivity limited- B cells - IGH V mutation
• Clonality is not equivalent to malignancy
• Interpret results in context
• IGH and TCRG rearrangements are not
markers of lineage
• Cross lineage rearrangements in lymphoid and
myeloid malignancies
Strategies for anti-cancer immunotherapy
118
Anti-neoplastic drugs
•
Alkylating agents (cisplatin, cyclophosmamide etc.)
– interact directly with cellular DNA, form bonds with nuclic acids
and proteins
•
Antimetabolites (methotrexate, fluorouracil, gemcitabine etc.)
– resemble cellular metabolites (folic acid, purine, pyrimidine)
– interfere with DNA precursors & cellular metabolism
•
Antitumor antibiotics (bleomycin, doxorubicin etc.)
– derived from soil fungus, some anti-infective activity
– interfere with DNA activity
•
Mitotic inhibitors (vinblastin, paclitaxel, docetaxel etc.)
– derived from plant extracts
– interfere with formation of mitotic spindle, arresting mitosis
•
Endocrine agents (tamoxifen, prednisolone, goserelin etc. )
– aromatase inhibitors, oestrogen antagonist, corticosteroids,
LHRH agonist
•
Molecularly targeted agents (retinoids, imatinib etc.)
– gene expression, monoclonal antibody, tyrosine kinase inhibitor
Sites of Action of Cytotoxic Agents
PURINE SYNTHESIS
•6-MERCAPTOPURINE
•6-THIOGUANINE
PYRIMIDINE SYNTHESIS
RIBONUCLEOTIDES
•METHOTREXATE
•5-FLUOROURACIL
•HYDROXYUREA
•PEMETREXED
DEOXYRIBONUCLEOTIDES
ALKYLATING AGENTS
AKYLATING LIKE
(INTERCALATING)
ANTIBIOTICS
DNA
•CYTARABINE
•GEMCITABINE
ETOPOSIDE
RNA
TOPOISOMER
PROTEINS
L-ASPARAGINASE
VINCA ALKALOIDS
ENZYMES
MICROTUBULES
TAXOIDS
Acknowledgement
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