Download Document

STUDY OF CANCER
& Immune System LS601
Professor Swapan K. Ghosh
[email protected]
237-2416
Lectures From April 4-15,2003
Fact Sheet:
 In 1996 554,740 deaths due to cancer in US alone.
 Over 8 million cancer patient in US are alive
 5 million of those were diagnosed 5 years ago.
 One of approx. 3 will experience cancer
“CANCER IS A CRIMINAL-IT DOES NOT FOLLOW
RULES”
Dr. S. Otani
CANCER: The term refers to 100 forms of the
disease.
Almost every tissue can get transformed into
malignancy. Caused by :
1. Uncontrolled growth by a cell that constitutes
the tissue;
2. The resulting impact on normal functioning of
body organs and disruption.
3. The ability of the aberrant cell to migrate to a
distant site and invade neighboring tissues,
a phenomenon called METASTASIS
.
Overview of cancer and
carcinogenesis
• Biology of cancer:
– Microevolutionary process
• Different stages of cellular
transformation
• Basic properties in cancer cells
• Etiology: aberrant natural selection
• Molecular genetics of cancer: Oncogenes,
Tumor suppressor genes
Microevolutionary processes
leading to cancer
• Animal bodies represent a society or ecosystem
– Individual members are cells that reproduce and
organize into assemblies or tissues
– Involves cell births, deaths, territorial boundary,
population sizes, species propagation
– BUT virtually no competition for survival and
all cells collaborate and sacrifice to produce
germ cells ensuring propagation (All somatic
cells die leaving no progeny)
1. Natural Selection in cells may
disturb collaboration & predispose
to cancer
2. Mutation, Competition and
natural selection among somatic
cells create a microevolutionary
environment promoting growth
of selfish aberrant cells or cancer
SPECIFIC TOPICS
General characteristics, classification, and
nomenclature of tumors
Properties: How cancer arises, How cancer spread
Grading and staging
Immunity and cancer
Therapy: surgery, radiation, chemotherapy and
biotherapy (immunotherapy)
Classification:
Carcinoma: cancer of epithelial tissues
Adenocarcinoma: cancer of glandular tissue; spread through
lymphatics
Sarcoma: cancer of stromal or mesenchymal layers of organs; spread
via blood.
Carcinosarcoma: mixtures of cancer cells from both epithelia and
mesenchma.
Teratoma: cancer of stem cells.
Undifferentiated neoplasms: poorly undifferentiated.
Terms and Definitions
Neoplasia : New aggressive growth of cells and
tissues putting pressure on neighboring tissues
(Causing abnormal swelling or tumor) or
invading neighboring tissues (cancer)
Hyperplasia: Means too much cell proliferation
or mitosis. This is abnormal but not cancer
Dysplasia: A cell is not only proliferating
excessively, but attains abnormal and orientation;
Pre-cancerous
More TERMS:
Metaplasia: conversion of one cell type into another,
such as, stratified oesophagus or lung tissues (due to
acidity or cigarette).The process is reversible and is not
cancer, but may lead to cancer.
Metastasis: Spreading to distant sites.
- First site where cancer is detected is called
primary site and the second site, secondary site.
-Small clumps of cancer cells (emboli) Spread by
migration through blood (called blood-borne or
hematogenous) or through lymphatics (lymphogenous).
-Cancer cells spread because they lose their
molecular address where to go.
More Jargons!
Anaplasia: Primitive undifferentiated state of cell
growth.
Aplasia: A loss of normal appearance and
disorganizations of tissues.
In situ cancer: abnormal growth at a particular site but
no invasion of neighboring tissues. Benign and fibrous.
Invasive cancer: Lethal and malignant as neighboring
tissues are invaded.
Characteristics of cancer cells
1. Infiltration and destruction of surrounding
tissues.
2. Loss of contact inhibition of
growth. Anchorage independent and aberrant
chromosome numbers or aneuplody
3. Variation in shapes and sizes based on degree
of differentiation.
4. Uncontrolled mitosis or cell proliferation or
growth rate. Less dependent on growth factors
5. Often migration to distant sites and loss of
similarity with parent tissues.
Cancer classification
• Sporadic cancer:
cancer without a
family history; nonhereditary and not
affecting off-springs
Mutations not present
in the germline cells.
Colon cancer mostly
sporadic
• Hereditary cancer:
– Mutations are present
in the germline cells
and predispose to
inheritance towards
developing cancer
(familial). Breast
cancer is an example
Most Cancers originate from a
single abnormal cell (Clonal
origin)
• Cancer is essentially a genetic disease but not, in
most cases, an inherited disease
• Cancer is nearly always of clonal origin
• Multiple mutations (at least TWO-HIT theory as
proposed by Knudson) in cancer cells
• Same genes are often altered by carcinogens,
radiation, viruses undergo translocation,
amplification or deletion
Cancer cells differently from
normal cells
Experimental evidence of cancer
clonality
•If cancer originates from one mutated cell, it could be
traced by looking at fingerprints of genes and/orproducts
•Tracking the identity of a marker X-chromosome that is
inactivated in cancer cells in females
•Studying chronic myelogeneous lymphomas (CML) and
tracking Ph chromosome as markers
•Studying Myelomas in which Myc oncogene has
translocated only to a specific Ig gene
X-Chromosome inactivation in
Females
•Only one of the two X chromosomes
inherited are inactivated in a cell.
• If cancer is monoclonal origin, it would
clearly have only one X-chromosome
inactivated.
•No mixed inactivation be seen in any
given tumor
How To Demonstrate Clonal
Origin of Cancer Cells
MYC GENE TANSLOCATION IN
MYELOMAS
Karyotype of a breast cancer cell
depicting translocations (DIFFERENT
COLORS)
Abnormal cells in Pap smear
What causes cancer
Various factors Contribute to the cause of cancer.
Carcinogens, that is, cancer producing agents can be
of physical, chemical, or of biological nature.
•Physical: Radioactivity, UV radiation, X-rays.
•Chemicals: from cigarette smokes, and shoots of
chimney or coal: hydrocarbon (benzpyrene),
DMBA, aromatic amine (in many synthetic dyes).
•Dietary factors: such as saturated fats, food
additives, lack of fruits and vegetables (antioxidants), alcohol, excess meat products
Mutations by carcinogens
What causes cancer
•Biological factors: hormones and viruses:
estrogen and pituitary hormones
promotes breast tumor.
•Oncogenic viruses: leukemia viruses
(Rous sarcoma virus) , Epstein-Barr
virus (Burkitt’s lymphoma), Papilloma
virus, Mammary tumor virus, Simian
virus
How Cancer Arises
1. Cancer cells violate the civic rules that govern normal
cells by not responding to go-signals for proliferation and
stop-signals for reproduction.
2. Cancer cells descend from a common ancestral cell:
clonal origin. But at some point one of the off-springs mutate
that becomes worse with more mutation, and finally the
accumulated mutated cells disobey all civic controls of normal
cells in a tissue, becoming invasive and malignant.
3. Since mutations occur at the gene level, that is, DNA
molecules that reside in the nuclei of the cells, most
human cancer can be traced there.
Proto-oncogenes from neighboring cells produce growth factors
that encourage cell growth during cell cycle by producing growthstimulatory signals. Mutation in these genes may cause cells divide
without any signal from outside. One example is mutated ras gene.
A quarter of all human tumors have mutated ras gene. Similarly
myc gene family if abnormal causes leukemia, lymphomas.
Receptors on the recipient cells that bind proto-oncogene growth
factors may also mutate and stimulate cell growth.Thus, in breast
cancer, Erb-B2 receptors behave abnormally.
Tumor suppressor genes that control unrestricted growth of
cells and inhibit cell growth. If they stop working, cancer cells grow
wild and uninterrupted. Example, pRB, P53 (tumor-suppressor
gene protein), TGF-beta (inhibits cell growth)
Cell cycle clock malfunction : Cyclin protein binds to
cyclin kinase (CDK) and releases the braking action of p53 on
cell proliferation from G1 to s phase. In cancer p53 is
inactive, so cells keep growing.
Apoptosis or programmed cell death. Normally, if a cell
is abnormal p53 gene product will promote suicide of the cell
thus avoiding cancer. If p53 does not function the abnormal
gets to live and be cancerous.
Cancer cells may Overcome p53’s grip on cell cycle by
producing excess of Bcl-2 protein that counteracts the action of
p53
All these make cancer cells immortal and cancerous.
How Cancers Spread
Cancer cells are malignant cells. What is
malignancy? It means invasiveness.
If a tumor does not disturb or infiltrate into
neighboring cells, they are BENIGN tumors.
But if they invade, they become MALIGNANT
and cancerous. AND IF They spread they
become metastatic cancer.
Invasion of neighboring cells and tissues:
1. Cancer cells lose their area code. It means that they do
not have glue or adhesion molecules with which to attach to
specific cells and form organs.
2. Cancer cells invade adjoining tissues by releasing
enzymes, called metalloproteinases, that dissolve basement
membranes and other extracellular matrix.
Once detached from where the cancer cell belongs, and
destroying the surrounding matrix, it goes into blood vessels
or lymphatics, gets carried to distant sites, and establish
metastatis.
Invasion and
metastasis
Cancer
Genetic
Initiation
(Oncogenes/ tr suppressor genes)
Epigenetic
Promotion
Progression
(clonal expansion)
Mitogenesis
Immune surveillance
Angiogenesis
TUMOR SUPPRESSOR GENES AND CANCER
Mutation 2
Mutation 1
Normal
X
Heterozygous
Other: Gene Methylation
Expression Levels
X
X
X
Loss of
Heterozygosity
Cell cycle
Quiescence = Go
DNA replication
S phase
R
Mitogens
Nutrients
Growth factors
G2
G1
Mitosis
Chromosome condensation and
segregation
General Cell Cycle Facts
Cell cycle is a fundamental cellular process
Wonderful convergence of cell biology, biochemistry and genetics
Principles are conserved in xenopus, yeast and mammals
G1
S
G2
M
Go
-restriction point; start
-DNA replication
-"rest" phase
-mitosis
-quiescence
Can be variable lengths.
Mammalian cells in culture: 14hr.
Liver cells: 1 yr (except under regeneration)
Kinase Activity
A Kinase Machine
Cyclin D1
CDK 4
G1
Cyclin E
CDK 2
Cyclin A
CDK 2
S
Cyclin B
CDC 2
G2/M
Cell cycle clock malfunction : Cyclin protein binds to
cyclin kinase (CDK) and releases the braking action of p53 on
cell proliferation from G1 to s phase. In cancer p53 is
inactive, so cells keep growing.
Apoptosis or programmed cell death. Normally, if a cell
is abnormal p53 gene product will promote suicide of the cell
thus avoiding cancer. If p53 does not function the abnormal
gets to live and be cancerous.
Cancer cells may Overcome p53’s grip on cell cycle by
producing excess of Bcl-2 protein that counteracts the action of
p53
All these make cancer cells immortal and cancerous.
Signaling Networks in Cancer
Fig. 2
Hanahan and Weinberg
Cell 100:57
Oncogenes
 Gene encoding proteins that is essential for the
initiation, promotion and progression of the
malignant state
 >100 different oncogenes
 discovered as the transforming genes of RNA
tumour viruses
 Proto-oncogene counterpart of viral-oncogene
- normal cellular genes which encodes proteins to
regulate cellular response to external stimuli that
controls cellular proliferation and differentiation.
- activated by mutation
- nomenclature
Classification of oncogenes
1. Growth factors - sis, ist
2. Growth factor receptors (RTK) - erb B2, fms
3. Non receptor tyrosine kinases - abl, src
4. GTP binding - ras
5. DNA damage repair - ATM, MSH2, B cl2
6. Serine/ threonine kinases
7. Nuclear binding - Myc, fos, jun
8. Misc - cell surface APC/ DCC
Tumor suppressor gene and
Oncogenes exert opposite effects
Proto=oncogenes and Suppressor genes
activated by DNA damage
How Rb controls cell cycle
Conversion of a proto-oncogene to
an oncogene
Proto-Oncogene products
A model for p53 function
RB gene mutations lead to cancer
Mechanisms of Proto-oncogene activation
1. Mutations:
* Point mutations/deletions/insertions
- most characterized in human trs, frequent in Ras
* Causes constitutive activation of the signal
transducing function of Ras protein
2. Gene amplification
Results from several rounds of unscheduled DNA
synthesis occurring during a single cell cycle
- HSR, DM Her2 Breast cacinoma, n-myc neuroblastoma
3. Chromosomal re-arrangement
* chromosomal translocations; inversions
* Gene activation -transcriptional activation
of Proto-oncogenes
eg. follicular lymphoma t (14:18) Bcl2
Burkitts lymphoma t (8:14) c-myc
* Gene fusion: codes for chimeric protein
eg. CML t (9:22) BCR/Abl.
APML t(15:17) PML/Rar
Ras - super gene family
– a group of G-proteins
– contains >50 proteins
– H-ras, K-ras, N-ras
– N-ras mutation most common alteration seen in
cancer
– Prenylation - Farneysl transferase
Ras activation in Leukemia
CMML - 32-65%, AML - 25-44%, ALL 6-18%
* Other mechanisms of activation
Y
SOS
G GRB2 adaptor mol.
S
P
G
S
R
Raf (MAPKKK)
GTP
GDP
MAPKK
MAPK
Ras - G protein system
Fos/ Jun activation
Cyclin D1
Ras GAP
Tumour suppressor genes:
* recessive genotype
- exception p27kip1
* Controls cell proliferation
*
>20 tumor suppressor genes
* Inherited cancers: Colon cancer
Retinoblastoma, Wilm’s tr, breast cancer
Neurofibromatosis,Li-Fraumeni syndrome,
Xeroderma pigmentosum
p19
p18
Growth factors
Ras, Raf, Myc
Fos, Jun
p27
p15
Radiation
DNA damage
Chemicals
Ckd 2,4,6
CD
1,2,3
E2F
p107
G1
E2F
p21
PCNA
CE
Cdk2
Rb
CA
P
p107
M
S
G2
Cell cycle
Cdk2
p53
DNA damage
MDM2
p53
P21
Bax/Bcl2
Waf1/ Cip1
Apoptosis
G1/S
Cyclin D/ cdk
R
Rb
P
pRB + E2F
S
G1
E2F
G2
Cell cycle damage and control
M
Angiogenesis
Proangiogenic
- Angiogenic growth factors
VEGF, FGF, TNF, HGF, IGF,TGF, Proliferin
Anti angiogenetic - Thrombospondin -1, 2
Angiostatin
Endostatin
Platelet factor 4
Apoptosis pathways
Caspase 9
Apaf 1
Apoptotic
signal
Caspase 3
Cyt C
Bcl2
AIF
Apoptosis
..
Cyt C
N
nucleus
Mitochondria
Bax
p53
G1
Viral Oncogenes Induce Proliferation
and Suppress Apoptosis
Adenovirus E1A
HPV E7
SV40 Lg T
RB
S
p53
APOPTOSIS
Adenovirus E1B (19K)
Bcl2-like
Adenovirus E1B(55K)
HPV E6
SV40 Lg T
pRB Pathway
Mitogenic Stimuli
(e.g. GF, Ras)
RB
E2F
D-Cyclin
CDK 4/6
E2F
P16
Ink4a
X
DNA Pol
Cyclin E, p19
DHFR, MYB
PPP
RB
Tumor Suppressor Genes
RB, p16
Oncogenes
Cyclin D1
From Sharpless and DePinho (1999)
Current Opinions in Genetics and Dev. 9:22
SENSOR of DNA DAMAGE
p53
increased levels
phosphorylation
conformational change
oligomerization
Transcriptional Activation
Cell Cycle
Repair
Apoptosis
p21
mdm2
GADD45
BAX
Figure 1
Levine (1997)
Cell 88:325
P53 and RB Crosstalk
p53
Ink 4
p19
p16
Bax
E2F1
mdm2
CyclinD
CDK4
RB
BCL2
Caspases
E2F1
Apoptosis
S-Phase
Apoptosis
p19
Similarity of Cell Cycle and Apoptotic Machinery
Apoptosis
Cell Cycle
A protease machine
A kinase machine
Caspases
CDKs
(Cysteine protease
which cleaves after ASPartate )
(Cyclin-dependent Kinase)
Inactive pro-enzyme
Activated by proteolytic processing cascade
Blocked by IAP (inhibitors of apoptosis)
Inactive
Activated by kinases, phosphatases
Blocked by CDKI
Evolutionarily Conserved
Evolutionarily Conserved
Ways to Score Apoptosis
Chromatin and Nuclear Condensation
Morphology on Hoechst dye immunofluorescence
Normal: round nuclei.
Apoptotic: irregular and condensed
DNA fragmentation (most often employed for apoptosis)
Appearance of a DNA ladder (due to intranucleosomal degradation)
Sub G1 population in FACS
TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling)
Membrane changes (early events)
Annexin binding
binds phosphatidylserine
generally on the inside of the plasma membrane, but flips
Cleavage of PARP by Caspases
Intrinsic Apoptosis Pathway
Figure 3
Wang, X. (2001)
GenesDev. 15:2922
Bcl2 gene Family
bcl-2 was identified as a gene that was
translocated in B-cell lymphomas.
--Unlike most oncogenes, bcl 2 extended cell
survival, rather than promote G1.
-Withdrawal of survival factors (e.g. IL-3)
leads to apoptosis, but bcl-2 protects.
Bcl2 Family of Apoptotic Regulators
Both pro- and anti-apoptotic members.
Anti-apoptotic: Bcl2, BCL-X-L
Pro-apoptotic: BAX , BAD
All have bcl-2 homology region (BH1-3)
All interact with each other.
e.g. BAX/bcl2, bcl-x ratio is a determinant of death
All affect mitochondrial membrane permeability.
Anti-apoptotic members block cytochrome c and SMAC release.
G1
G0
Jun, FOS
p53
M
Cyclin D
CDK4
p16 Ink 4a
G2
p21
Cyclin E
CDK2
E2F RB
Cyclin A
CDK2
DP
PP PP
E2F DP
(inactive E2F)
PPP
S
RB
E2F DP
(active E2F)
Tissue
Differentiation
Intrinsic and Extrinsic Death Pathway
Extrinsic
Intrinsic
Figure 2
Johnstone et al
(2002) Cell 108:153
Treatment approaches in Oncology
Surgery
Radiotherapy
Chemotherapy
Biotherapy (experimental):
Immunotherapy (magic bullet)
Gene therapy
Bone marrow transplantation
How do you select treatment modality?
After pathological determination of the tumor
type, and TUMOR STAGING
What is tumor staging? Categorizing malignant
tumors relative to their potential for invasiveness
and metastatic capability. Stage I = tumor at the
primary site; Stage II & III= moderate spread and
invasion;
Stage IV= extensive spread and invasion (poorly
differentiated, pleomorphic, visible mitoses; least
chance of survival).
Another way of tumor grading: TNM
system where T means how big the
tumor is at the primary site;
N means enlargement of regional
lymph node; M means extent of
metastasis. hat does ToNoMo mean?
Surgery: good for benign tumor, less for
malignant tumor depending on the stage of
the tumor. For invasive tumor removal of
lymph node may be necessary.
Raises
concern for iatrogenic spread.
Radiotherapy: Destructive dose of ionizing
radiation from cobalt-60 may kill tumors.
Since it raises temperature, it causes burn in
skin other non tumor areas. Not all tumors are
radiosensitive.
Chemotherapy: Interferes with metabolism
or mitosis of tumors. Not always work.
Harmful for normal tissues too. Often tumors
develop resistance.
(MDR expression)
Combination therapy: Radiation + Chemo.
As well as a combination of various drugs.
Immunotherapy : potentially the best
treatment.
Immunotherapies:
biological response modifiers
(Cytokines), anti-tumor antibody, anti-tumor antibodytoxin conjugates (magic bullet), LAK (lymphokineactivated killer) cells, Adjuvants.
Gene Therapy:
Inserting missing genes into cancer cells
Inserting anti-oncogene DNA
Inserting tumor-supressor genes such as p53, pRB
DNA vaccine
Figure 3. The cell cycle clock and cancer. The cell cycle clock-composed of an assembly of interacting proteins in the nucleus-normally integrates messages from the stimulatory and inhibitory
pathways and, if the stimulatory messages win out, programs a cell's
advance through its cycle of growth and division. Progression
through the four stages of the cell cycle (a) is driven by rising levels
of proteins called cyclins: the D type, followed by E, A and B. A
crucial step in the cycle occurs late in G1 at the restriction point (R),
when the cell decides whether to commit itself to completing the
cycle.
For the cell to pass through R and enter S, a molecular "switch" (b)
must be flipped from "off" to "on". As levels of cyclin D cyclin E
rise, these proteins combine with and activate cyclin-dependent
kinases (1). The kinases grab phosphate groups (2) from
molecules of ATP (adenosine triphosphate) and transfer them to
protein pRB, the master brake. When pRB lacks phosphates, it
actively blocks cycling (and keeps the switch in the "off" position)
by sequestering other proteins termed transcription factors. But
after the cyclin-kinase complexes add enough phosphates to pRB,
the brake stops working (3); it releases the factors, freeing them to
act on genes. The liberated factors then spur production of various
proteins required for progression through the cell cycle.
In c, the switch is placed in the larger context of the many
molecular interactions that regulate the cell cycle. Flipping the
switch to "on" can be seen above the R point. Over-activity of the
stimulatory proteins cyclin D, cyclin E and CDK4 have been
implicated in certain human cancers. Inactivation of various
inhibitory proteins has also been documented. The affected
proteins include p53, pRB, p16 and p15.
Mitochondria in apoptotic signaling
Clinical Applications
1. Diagnosis - oncogenes c-myc in Burkitts
Bcr/Abl CML
2. Detection of MRD
3. Prognosis
Bcr/Abl CML
n-myc neuroblastoma
Her 2 breast cancer
4. Predictive oncology
MEN Type II, Li-Fraumeni syndrome
Retinoblastoma, Wilms tr, Breast ca.