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STUDY OF CANCER & Immune System LS601

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Title: STUDY OF CANCER & Immune System LS601


1
STUDY OF CANCER Immune System LS601
  • Professor Swapan K. Ghosh
  • Lsghosh_at_scifac.indstate.edu
  • 237-2416
  • Lectures From April 4-15,2003

2
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
3
  • CANCER The term refers to 100 forms of the
    disease.
  • Almost every tissue can get transformed into
    malignancy. Caused by
  •  
  • Uncontrolled growth by a cell that constitutes
    the tissue
  • The resulting impact on normal functioning of
    body organs and disruption.
  • The ability of the aberrant cell to migrate to a
    distant site and invade neighboring tissues,
  • a phenomenon called METASTASIS
  • .   

4
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

5
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)

6
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

7
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)  

8
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.
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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
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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. . .
13
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.
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  • Characteristics of cancer cells
  • Infiltration and destruction of surrounding
    tissues. 
  • Loss of contact inhibition of growth. Anchorage
    independent and aberrant chromosome numbers or
    aneuplody
  • Variation in shapes and sizes based on degree of
    differentiation. 
  • Uncontrolled mitosis or cell proliferation or
    growth rate. Less dependent on growth factors
  • Often migration to distant sites and loss of
    similarity with parent tissues.

16
Cancer classification
  • Sporadic cancer
  • cancer without a family history non-hereditary
    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

17
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

18
Cancer cells differently from normal cells
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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

21
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

22
How To Demonstrate Clonal Origin of Cancer Cells
23
MYC GENE TANSLOCATION IN MYELOMAS
24
Karyotype of a breast cancer cell depicting
translocations (DIFFERENT COLORS)
25
Abnormal cells in Pap smear
26
  •  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
    (anti-oxidants), alcohol, excess meat products

27
Mutations by carcinogens
28
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 (Burkitts lymphoma),
    Papilloma virus, Mammary tumor virus, Simian
    virus

29
  • How Cancer Arises
  • Cancer cells violate the civic rules that govern
    normal cells by not responding to go-signals for
    proliferation and stop-signals for reproduction.
  •  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.

30
Proto-oncogenes from neighboring cells produce
growth factors that encourage cell growth during
cell cycle by producing growth-stimulatory
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)
31
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 p53s 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.  
32
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.
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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.
36
Invasion and metastasis
37
Cancer
Genetic (Oncogenes/ tr suppressor genes)
Initiation Promotion
Epigenetic (clonal expansion)
Mitogenesis Immune surveillance Angiogenesis
Progression
38
TUMOR SUPPRESSOR GENES AND CANCER
Mutation 2
X
Mutation 1
X
X
X
Loss of Heterozygosity
Heterozygous
Normal
Other Gene Methylation Expression Levels
39
Cell cycle
Quiescence Go
DNA replication
S phase
R
G2
G1
Mitogens Nutrients Growth factors
Mitosis
Chromosome condensation and segregation
40
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 -restriction point start S -DNA
replication G2 -"rest" phase M -mitosis
Go -quiescence Can be variable lengths.
Mammalian cells in culture 14hr. Liver cells
1 yr (except under regeneration)
41
A Kinase Machine
Cyclin B CDC 2
Cyclin D1 CDK 4
Cyclin E CDK 2
Cyclin A CDK 2
Kinase Activity
G1
S
G2/M
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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 p53s 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.  
45
Signaling Networks in Cancer
Fig. 2 Hanahan and Weinberg Cell 10057
46
  • Oncogenes
  • Gene encoding proteins that is essential for the
    initiation, promotion and progression of the
    malignant state
  • gt100 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

47
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
48
Tumor suppressor gene and Oncogenes exert
opposite effects
49
Protooncogenes and Suppressor genes activated by
DNA damage
50
How Rb controls cell cycle
51
Conversion of a proto-oncogene to an oncogene
52
Proto-Oncogene products
53
A model for p53 function
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RB gene mutations lead to cancer
56
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

57
3. Chromosomal re-arrangement chromosomal
translocations inversions Gene activation
-transcriptional activation of
Proto-oncogenes eg. follicular lymphoma
t (1418) Bcl2 Burkitts lymphoma t (814)
c-myc Gene fusion codes for chimeric protein
eg. CML t (922) BCR/Abl. APML
t(1517) PML/Rar
58
Ras - super gene family
  • a group of G-proteins
  • contains gt50 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

59
R
S
G
Y
P
SOS
S
Raf (MAPKKK)
Ras GAP
GRB2 adaptor mol.
G
GTP
MAPKK
GDP
MAPK
Ras - G protein system
Fos/ Jun activation
Cyclin D1
60
Tumour suppressor genes recessive genotype
- exception p27kip1 Controls cell
proliferation gt20 tumor suppressor genes
Inherited cancers Colon cancer Retinoblastoma,
Wilms tr, breast cancer Neurofibromatosis,Li-Fra
umeni syndrome, Xeroderma pigmentosum
61
Radiation DNA damage Chemicals
p19
Growth factors Ras, Raf, Myc Fos, Jun
p18
p27
p15
Ckd 2,4,6
PCNA
p21
p53
C D 1,2,3
p107
G1
E2F
C E
Cdk2
E2F
Cdk2
Rb
C A
p107
P
M
S
G2
Cell cycle
62
DNA damage
MDM2
p53
Bax/Bcl2
P21 Waf1/ Cip1
Apoptosis
G1/S
Cyclin D/ cdk
R
pRB E2F
Rb
P
S
G1
E2F
G2
M
Cell cycle damage and control
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Angiogenesis Proangiogenic - Angiogenic
growth factors VEGF, FGF, TNF, HGF, IGF,TGF,
Proliferin Anti angiogenetic - Thrombospondin
-1, 2 Angiostatin
Endostatin Platelet
factor 4
66
Apoptosis pathways
Caspase 9
Apaf 1
Caspase 3
Cyt C
Apoptotic signal
Apoptosis
Bcl2
.
.
AIF
Cyt C
Bax
N
nucleus
p53
Mitochondria
67
Viral Oncogenes Induce Proliferation and
Suppress Apoptosis
G1
Adenovirus E1A HPV E7 SV40 Lg T
RB
S
68
pRB Pathway
Mitogenic Stimuli (e.g. GF, Ras)
RB
X
E2F
D-Cyclin CDK 4/6
DNA Pol Cyclin E, p19 DHFR, MYB
E2F
PPP
P16 Ink4a
RB
Tumor Suppressor Genes RB, p16 Oncogenes Cyclin
D1
From Sharpless and DePinho (1999) Current
Opinions in Genetics and Dev. 922
69
SENSOR of DNA DAMAGE
p53
increased levels phosphorylation conformational
change oligomerization
Transcriptional Activation
Cell Cycle p21 mdm2
Repair GADD45
Apoptosis BAX
Figure 1 Levine (1997) Cell 88325
70
P53 and RB Crosstalk
p53
Ink 4
p16
p19
Bax
mdm2
CyclinD CDK4
RB
E2F1
BCL2
Caspases
E2F1
Apoptosis
Apoptosis
S-Phase
p19
71
Similarity of Cell Cycle and Apoptotic Machinery
Apoptosis A protease machine Caspases (Cystein
e protease which cleaves after ASPartate
) Inactive pro-enzyme Activated by proteolytic
processing cascade Blocked by IAP (inhibitors of
apoptosis) Evolutionarily Conserved
Cell Cycle A kinase machine CDKs (Cyclin-depen
dent Kinase) Inactive Activated by kinases,
phosphatases Blocked by CDKI Evolutionarily
Conserved
72
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
73
Intrinsic Apoptosis Pathway
Figure 3 Wang, X. (2001) GenesDev. 152922
74
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.
75
Bcl2 Family of Apoptotic Regulators
Both pro- and anti-apoptotic members. Anti-apoptot
ic 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.
76
Tissue Differentiation
G1
Jun, FOS
p53
M
p16 Ink 4a
p21
G2
RB
DP
Cyclin A CDK2
PPP
RB
S
PP
PP
DP
DP
(inactive E2F)
(active E2F)
77
Intrinsic and Extrinsic Death Pathway
Figure 2 Johnstone et al (2002) Cell 108153
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Treatment approaches in Oncology Surgery Radiot
herapy Chemotherapy Biotherapy (experimental) Imm
unotherapy (magic bullet) Gene therapy Bone
marrow transplantation
84
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).  
85
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?
86
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.
87
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.
88
Immunotherapies biological response modifiers
(Cytokines), anti-tumor antibody, anti-tumor
antibody-toxin conjugates (magic bullet), LAK
(lymphokine-activated 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
89
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.   
90
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.
91
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.
92
Mitochondria in apoptotic signaling
93
Clinical Applications
1. Diagnosis - oncogenes c-myc in Burkitts
Bcr/Abl CML 2. Detection of
MRD Bcr/Abl CML 3. Prognosis n-myc
neuroblastoma Her 2 breast cancer 4.
Predictive oncology MEN Type II, Li-Fraumeni
syndrome Retinoblastoma, Wilms tr, Breast ca.
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