Categories of genes targeted during

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Categories of genes targeted during multistage
carcinogenesis
Intracellular Circuitry Agonist-induced signal
transduction DNA replication and repair
Cell cycle control Cell fate survival,
differentiation, senescense, and
apoptosis Cell surface and extracellular
functions Adhesion molecule proteases
angiogenic factors, etc
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Cyclins and Cyclin-Dependent Protein Kinases (CDK)
Activate Protein Kinase
cdc2CDKcyclin-dependent protein kinase.
There are multiple cyclins and CDKs. Specific
cyclins are made and degraded at different
stages of the cell cycle. Activity is further
modulated by phosphorylation/dephosphorylation,
nuclear translocation, association with other
proteins, and proteolysis.
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D11S46 BCL1 CCND1/PRAD1 EXP1 HST1/FGF4 INT2/FGF3
EMS1 D11S833E
0kb 120kb 600kb 1000kb
11p 1 1 11q
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11q13 Amplicons
15Mb
Diagram of Human Chromosome 11q13 Amplicons
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Increased Expression of Cyclin D1 in Human
Cancers

• Breast 60
• Prostate 24
• Head and Neck, Esophagus 40
• Stomach, Small Bowel, Colon
• Lung (NSCLC), Bladder, Liver, 10-13
• Parathyroid Adenomas, Lymphoma

Especially ERtumors Centrocytic
lymphomas Also occurs in precursor lesions
(Barretts, polyps, CIS)
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Mechanistic Studies on Cyclin D1

• Overexpression enhances cell transformation
  and gene amlification in transgenic mice causes
  mammary tumors or esophageal dysplasia
• Antisense inhibits tumor phenotype and
  tumorigenicity (in 4 types of cancer cells)
• Binds to and stimulates the ER, but inhibits
  the AR
• Expression controlled by APC/b-catenin/TCF
  pathway

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Polymorphism of Cyclin D1

• There is an A/G polymorphism in splice donor site
  of exon 4 which influences ratio of two
  transcripts, a and b
• 2. Two transcripts may have different functions
• In NSLC GG genotype correlates with good
  prognosis
• In SCCHN GG genotype correlates with poor
  prognosis

Betticher, et al.,Oncogene 111005,
1995 Matthias, et al., Clin. Cancer Res. 4,
2411, 1998
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Cyclin E Overexpression and dysregulation
frequent in various human cancers, usually in
absence of gene amplification or rearrangement.
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Principles of Cdk Regulation
Cyclin
14T
T
Cdk
15Y
KAP
Wee1
CAK
CDC25
Active
CKI
Cyclin
14T
P
T
Cdk
15Y
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CDC25A and B are Potential Oncogenic Proteins

• CDC25A and B
• are induced and activated in virally
  transformed cells
• interact with and activated by Raf1 in vitro
• are down-stream targets of the oncogene c-myc
• can cooperate with activated Ras and Rb-/- to
  transformed cells.
• are overexpressed in several types of human
  cancer

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Summary

• Ectopic Cdc25B mRNA was readily detected in
  the lactating and non-lactating mammary glands of
  transgenic mice.
• High levels of Cdc25B protein were detected in
  the lactating
• mammary glands of transgenic mice.
• MMTV-Cdc25B mice did not develop spontaneous
  mammary tumor.
• MMTV-Cdc25B mice were more susceptible to
  DMBA-induced mammary tumor formation.

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p27Kip1 is associated with poor prognosis in
breast, colon, stomach, prostate and oral
cavity carcinomas non-small cell lung cancer,
gliomas endocrine tumors, lymphomas also
seen in some precursor lesions due to
proteasome-mediated proteolysis Readily scored
for by immunostaining
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Inhibitors of Cyclin/CDK Activity

• Antisense to cyclin D1
• Staurosporine and UCN-01
• Butyrolactone
• Flavopiridol and L86876
• Olomoucine and Rosovitine
• 9-hydroxy-ellipticine
• Suramin
• Isopentenyladenine
• p16 derived peptides
• In clinical trial
• Note Limited kinase specificity p53-independent
  apoptosis inducing effects

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Paradoxical increases in the expression of tumor
suppressor genes in cancer cells p27, pRB, p21,
p16
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• Evidence for Positive Feedback Loop
• Between Cyclin D1 and p27
• Concerted overexpression in cancer cell lines and
• primary cancers
• Cyclin D1 p27
• 3. Cyclin D1 p27
• Similar evidence indicates cyclin E p27
  
• Feedback loops are cell-type specific

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Negative Feedback Control of Cyclin D1 in Mammary
Epithelial Cells
Cyclin D1 G1
S p27Kip1
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Overview

• Mutistage carcinogenesis involves numerous
  genetic and epigenetic abnormalities in gene
  expression
• The genes that are targeted are components of
  interacting networks of signal transduction that
  display homeostatasis, whose phenotypic effects
  are highly context dependent.
• 3. Therefore, cancer cells have acquired bizarre
  patterns of cell circuitry-which might be
  exploited in cancer prevention and therapy.

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Clonal Evolution of Cancer

• Also epigenetic changes

• Progressive disorder in signal
  transduction balanced by
  homeostatic mechanisms

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?

• Since cancer cells have numerous genetic changes
  what is the effect of reversing only one of these
  changes

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Examples of Oncogene AddictionStudies in Mice

• Oncogene Type of Neoplam Reference
• c-myc T cell and acute myeloid Felsher and
  Bishop, 1999
• leukemia
• Bcr-Abl leukemia Huettner, et al., 2000
• H-ras melanoma Chin, et al., 1999
• k-ras lung Chin, et al., 1999
• Jackson, et al., 2001
• c-myc pancreatic b cell Pelangaris, et al.,
  2002
• Fisher, et al., 2001
• c-myc osteogenic sarcoma Jain, et al., 2002
• neu breast Moody, et al., 2002
• Wnt1 breast Grunther, et al., 2003
• Switching off indicated oncogene led to
  inhibition, differentiation,
• apoptosis and/or tumor regression

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Examples of Oncogene AddictionStudies in Human
Cancer Cell Lines

• Oncogene Carcinoma Cell Line Reference
• HER-2 breast Colomer, et. al., 1994
• Cyclin D1 esophageal Zhou, et al., 1995
• colon Arber, et al., 1997
• pancreatic Kornmann, et al., 1999
• squamous Sauter, et al., 1999
• K-ras pancreatic Aoki, et. al., 1997
• K-ras pancreatic Brummelkamp, et
  al., 2002
• __________________________________________________
  _________
• Treatment of these cell lines with an
  antisense oligonucleotide to the respective
  oncogene caused growth inhibition and in some
  cases decreased tumorigenicity or increased
  chemosensitivity
• used RNAi

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Gene Addiction

• Cancer cells that have increased activity of an
  oncogene are more dependent on that oncogene than
  normal cells
• Examples cyclin D1 overexpression activated
  K-ras HER-2 amplification

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Gene Hypersensitivity

• Cancer cells that have lost the expression of a
  tumor suppressor gene are more sensitive to the
  inhibitory effects of that gene than normal cells
• Examples Effects of reintroduction of APC, RB,
  or p53 into cancer cells

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Hanahan and Weinberg, 2000
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Gene addiction and hypersensitivity in cancer
cellsare exploitable targets for chemoprevention
and therapy
Weinstein, I.B. Science 297, 63-64, 2002
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1. How do we identify the addict?2. Escape
through mutation/altered circuitry3. Tumor
heterogeneity, between and within
tumors4. Pharmacologic problems
Oncogene Addiction Caveats
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Escape From Oncogene Addiction in a Mouse Model

• Induce mammary tumors in mice with c-myc
• Switch off c-myc expression
• 50 of tumors regress and 50 partially regress
• Tumors then recur that are
• c-myc independent
• some have activated k-ras oncogene
• others may have other 2o changes
• _______________________________________
• Boxer et al., Cancer Cell 6, 577-586, 2004.

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Oncogene Addiction Relevance to Molecular
Targeting in Cancer Therapy

• Disease Target Drug/Agent
• Breast Cancer ERa Tamoxifen
• Her2 Herceptin
• CML Bcr/Abl Imatinib
• (TK) (Gleevac)
• Lung Cancer EGFR Iressa,
• (RTK) Tarceva
• Kidney Cancer VEGFR Ligand Avastin
• VEGFR Kinase inhibitors
• __________________________________________________
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• Preferential for mutated EGFR present in a
  subtype of lung cancers.

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Abnormalities in Cell Cycle-Related Oncogenes in
Human Cancers
Gene Type of abnormality Type of
cancer Cyclin Cyclin A HBV insertion Liver
Cyclin E increased expression and low MW
forms Breast, colon, prostate Cyclin
D1 Chrosomal translocation Parathyroid
(PRAD1) B cell lymphoma (BCL1) Gene
amplification Breast, esophagus, head
neck, lung, liver, bladder Increased
expression w/o amplification Breast,
colon Cyclin-dependent kinase
CDK4 Amplification Gliomas,
sarcoma Mutation, disruption of p16
binding Melanoma Phosphatase
CDC25B Increased expression Breast
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Abnormalities in Cell Cycle-Related Tumor
Suppressor Genes in Human Cancers
Gene Type of abnormality Type of
cancer Cyclin-dependent kinase inhibitor
p16INK4a Deletion, point mutation Melanoma,
pancreas, DNA methylation esophagus,
gliomas, etc. p19Arf Deletion Melanoma
p15INK4b Deletion Leukemias, lung, etc.
p21CIP1 Impaired induction in p53-/- cells
Numerous types of cancer p27KIP1 Impaired
function in Numerous types of
cancer TGFb-resistant cells Increased
expression Esophagus, breast, colon G1/S
checkpoint control protein p53 Mutation or
virus inactivation Numerous types of cancer
Rb Mutation or virus inactivation Numerous
types of cancer