Retinoids and Cancer Prevention

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Retinoids and Cancer Prevention
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Outline Carcinogenesis Chemoprevention and
chemotherapy Retinoids Retinoid receptors In
vitro model Clinical study
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Multistage Carcinogenesis

• Carcinogen exposure
• Initiation
• Promotion
• Conversion
• Progression

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Inhibition of Carcinogenesisat the cellular level

• Modulation of the metabolism or
• detoxification of carcinogens
• Inhibition of initiated cell proliferation
• Inhibition of the expansion of premalignant
• clones
• Induction of differentiation
• Induction of apoptosis (of initiated cells)
• Inhibition of angiogenesis

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Chemoprevention

• Use of specific natural or synthetic chemical
  agents to reverse, suppress, or prevent the
  carcinogenic process.
• Reduce exposure
• -Inhibit carcinogenic initiation,
• promotion and progression
• Prevent progression
• -High risk individual (family history, second
• primary, etc)
• Decrease recurrence (primary site and
  metastasis)
• M. Sporn

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-Chemoprevention -Chemotherapy
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How does cancer prevention
work? Reversaldisappearance of a risk
phenotype (dysplastic cells) -Retinoids and
chemprevention Sporn-1976-1980 Suppressiondela
y occurrence -Retinoids (Cancer Res., 39,
3977-3980, 1979) -Tamoxifen (Craig
Jordan) Elimination (cure)permanent
protection -Nonsteroidal anti-inflammatory drugs
(NSAIDs) Colon-polyp regression (D. Alberts)
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Targets for Chemoprevention

• Healthy population at risk (eg. former smokers)
• Individuals with premalignancies
• Patients cured of cancer, facing high risk of
  recurrence

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Classification of chemopreventive agents

• Category of inhibitors Sequence leading to
  malignancy
• Precursor components
• Inhibitors preventing
• formation of carcinogens
• Carcinogenic compounds
• Blocking agents
• Reactions with cellular targets
• Suppressing agents
• 
  Expression of malignant properties

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all-trans retinoic acid
N-phenylretinamides
Chemical name R2 R3 R4 N-(2-hydroxyphenyl)ret
inamide OH H H N-(3-hydroxyphenyl)retinamide
H OH H N-(4-hydroxyphenyl)retinamide H
H OH N-(2-carboxyphenyl)retinamide COOH
H H N-(3-carboxyphenyl)retinamide H
COOH H N-(4-carboxyphenyl)retinamide H
H COOH N-(4-methoxyphenyl)retinamide H H CH3O
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Fundamental Processes Regulated by Retinoids at
the Cell Level
Cell proliferation Cell differentiation Apoptosi
s Motility and invasion Adhesion
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Steroid hormone receptor superfamily
P. Chambon
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Nuclear receptors share a common structure and
functional domains
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Retinoid Receptor Defects in Human Cancer

• Receptor
  Cancer
• RAR-?
  APL, Breast
• RAR-?
  HN, Lung, Esophagus, Breast,
• 
  Bladder,Prostate, Stomach,
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  Pancreas, Cervix, Kidney
• RAR-?
  Skin SCC, NSCLC, germ cell
• RXR-?
  DNS/melanoma, skin SCC
• RXR-?
  NSCLC
• RXR-?
  __

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RXR-heterodimeric receptors and biological effects
Receptors
Biological effects PPAR?
Fat catabolism PPAR?
Migration

differentiation PPAR?
lipid metabolism RARs/RXRs

Proliferation/differentiation VDR

Proliferation/differentiation TR

Proliferation/differentiation LXR/FXR
Cholesterol
metabolism CAR
androstane and xenobiotics PXR/SXR
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CHEMOPREVENTION STUDIES IN VITRO

• Identify new chemopreventive agents
• Identify intermediate end-point biomarkers
• Understand mechanisms of action of
  chemopreventive agents

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In vitro Cell Models for Screening
Chemopreventive Agents

• Normal cells
• - short term cultures (NHEK, NHBE,Pr Epi)
• Premalignant cells
• - SV40 T-immortalized cells
• - HPV-immortalized cells
• - Spontaneously-immortalized cells (NIKS,HaCaT)
• Transformed cells
• Immortalized cells exposed to carcinogen
• (C50, 3PC)
• Malignant cells
• - (SBR12-p9)

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Criteria for in vitro studies

• The nature of the cell type (Do the cells model
  tumor type?)
• whether primary cells, nontumorigenic or
  tumorigenic cells were used
• the appropriateness of controls
• toxic effects vs specific signaling events
• whether appropriate concentration range of agent
  was used
• the relevance of the endpoints including
  inhibition of mutagenesis, morphological
  transformation, anchorage-independent growth,
  cell-cell communication, differentiation, and
  apoptosis
• whether culture conditions mimic physiological
  setting (eg. organotypic culture)
• Most important Are the effects observed in-vitro
  representative of events in-vivo?

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Normal Human Skin
Cornified Layer
-dead cells
Granular Layer
Ca2
-metabolically
-Loricrin
-Keratins 1,10,13
active
-Filaggrin
-involucrin
-RAR
a/g
-
c/ebp
-cornifin
a
-RXR
a/b/g
Epidermis
Spinous Layer
-TG1
-junD
(squamous layer)
-RAR
b
-STAT1,2,3
-JunB
-Tyk2
-JAK1
-Fra2
-P48
-fosB
-Keratins 5, 8,
Basal Layer
RA
14,19
-mitotically active
-bcl-2
-IFNAR1,2
Basement Membrane
Dermis
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Raft Culture of Keratinocyte-derived Cells
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Human Skin Cell Organotypic Raft Culture
NIKS
HaCaT
SRB12-p9
200X
400X
normal
premalignant
SCC
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Relationship between differentiation,
proliferation and apoptosis and malignancy
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Apoptosis, carcinogenesis and chemoprevention
Apoptosis may be deregulated during
carcinogenesis. Clonal expansion of initiated
cells and the formation of premalignant lesions
may involve a decrease in apoptosis rate in
addition to an increase in proliferation. Some
tumor promoters inhibit apoptosis. Agents that
can induce or enhance apoptosis in premalignant
cells can exert chemopreventive effect on
carcinogenesis.

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HYPOTHETICAL PATHWAYS OF APOPTOSIS INDUCTION BY
4HPR
Antioxidants, bongkrekic acid
Mitochondria ROS/Dy
Apoptosis
Cyt. C casp. 9 casp. 3
ceramide? (Fumonisin B)
4HPR
Nucleus RARs/RXRs
Differentiation -Proliferation control
Antagonists
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Mechanisms of 4HPR-Induced Growth Inhibition/
Apoptosis (I)

• Downregulation of c-myc in prostate ca cells
  (PC3) (Igawa et al., Prostate, 24299, 1994)
• Upregulation of Rb protein in breast ca cell
  lines (MCF-7, T-47D (Kazmi et al., Cancer Res.,
  561056, 1996)
• Downregulation of cyclin D1, cdc2, and cdk4
  expression and Rb phosphorylation and increase in
  ceramide synthesis in HL60 cells (DiPietrantonio
  et al., Int. J. Cancer 7853, 1998 BBRC 224837,
  1996)
• Downregulation of PCNA, cyclins D and E, p34cdc2,
  p53 and Rb in androgen-independent prostate ca
  cell line JCA-1 (Hsieh et al., Biochem. Mol.
  Biol. Int., 37499, 1995)
• Downregulation of c-erb-B1 (EGF-R) in esophageal
  ca. cells (Muller et al., Cancer Lett., 11395,
  1997) and breast ca cells (Pellegrini et al.,
  Cell Growth Diff., 6863, 1995)
• Downregulation of c-erb-B2 (HER-2/neu) mRNA and
  protein in breast carcinoma cells (Pellegrini et
  al., Cell Growth Diff., 6863, 1995 Grunt et
  al., Br. J. Cancer 7879, 1998)

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Mechanisms of 4HPR-Induced Growth Inhibition/
Apoptosis (II)

• Upregulation of mac25, a putative tumor
  suppressing IGF-BP family member, in senescing
  mmmary epithelial cells (Swisshelm et al., Proc.
  Natl. Acad. Sci. USA 924472, 1995)
• Downregulation of IGF-I-like protein in medium,
  reduction in IGF binding proteins (IGF-BPs) 4 and
  5, decrease in type I IGF receptor mRNA and
  IGF-I binding to breast cancer cells (Favoni et
  al., Br. J. Cancer 772138, 1998)
• Decreased expression of androgen receptor in
  androgen-dependent prostate ca. cell line (LNCaP)
  (Hsieh and Wu, Prostate 3397, 1997)
• Increased TGF-b1 and TGF-b receptor type II in
  prostate carcinoma cells (Roberson et al., Cell
  Growth Diff., 8101, 1997)
• Decreased integrin expression and cell-matrix
  adhesion in neuroblastoma cells (Rozzo et al.,
  Int. J. Cancer, 70688, 1997)

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Cell cycle distribution following 4 days
treatment with 1mM retinoids
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Chemopreventive agents shown to induce apoptosis
in cancer cells-1
Agent Cells
Reference Green tea polyphenols Various tumor
cells Fujiki 1999 (epigallocatechin gallate,
epigallocatechin) Selenium (monomethylated
form) Various tumor cells Ganther,
1999 selenocystamine
and in vivo Stewart,
1999

el-Bayoumy, 1995 Tributyrin (a
triglyceride Mammary carcinoma Heerdt,
1999 analog of butyrate) Monoterpenes (limonene,
Various tumor cells Gould,
1997 perillyl alcohol) Crowell,
1999 Hybrid polar agents
Various tumor cells Marks, 1999 (HMBA, SAHA)


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Chemopreventive agents shown to induce apoptosis
in cancer cells-2

Cells induced Agent to
undergo apoptosis Reference N-(4-hydrox
yphenyl)retinamide Various tumor
Delia, 93 Retinoids
cells
Lotan, 95 Vitamin D analog EB1089
Prostate cells
Nickerson and

Huynh 99 Human chorionic
Breast epithelial cells Srivasstava,
98 gonadotropin (hCG) ATRA
Myeloma cell lines
Taetle, 96 ATRA, 13-cis RA
Breast carcinoma cells
Toma, 97 Genistein (soy flavone)
Breast carcinoma Eleanene (Chinese
medicinal Various tumor cells
Zheng, 97 herb constituent) Celecoxib
Colon
cancer cells
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Chemopreventive agents shown to induce apoptosis
in cancer cells-3

Cells induced Agent to
undergo apoptosis Reference
Sulindac, sulfone and sulfoxide Mammary
cells Thompson, et al 97

Sulindac derivatives
Prostate cancer
Lim 99 Silymarin
Epidermoid carcinoma Zi Agarwal 97


Aspirin
Gastric cancer cells Wong,
et al 99 Antiestrogen
Breast cancer cell lines Kandouz, et al
97 Progestins
Androgen
(dihydrotestosterone) 1,25(OH)2 vitamin D3
Prostate cancer Blutt
Weigel 99

DFMO
Colonic epithelium
Li, et al 99
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Chemopreventive agents shown to induce apoptosis
in cancer cells-4

Cells induced Agent to
undergo apoptosis Reference
DFMO
Esophageal cells
Fong, et al 98
in Zu 2 deficient
rats 9-cis-RA
Colonic epithelium Zheng, et
al 99

Resveratrol

Huang, et al 99 (skin of grapes)

Genistein (soy
isoflavone) Prostate cancer cells
Davis, et al 98

Soy isoflavones
Human bladder
Zhou, et al 98 (genistein, genistin,
daidzein cancer cells and biochanin A)
Prostate cancer cells
Onozawa, et al 98 Curcumin (from plant of
Human basal cell Tee, et
al 98 ginger family)
carcinoma cells Sulindac and sulindac
Human breast cancer Hau, et al
98 sulfone
cell lines Aspirin
Colon crypt cells
Barnes, et al 98
exposed
to carcinogen
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Mechanisms of action of chemopreventive agents
that induce apoptosis-a
Agent Mechanism (Reference) Tributyrin Increas
ed bax dissipation of MTP Release of
cytochrome C into cytosol, PARP
Cleavage (caspase-independent) (Heerdt, et al
99) Tea polyphenols Scavenging of reactive
oxygen species (Lin, et al 99) Vitamin
1,25(OH)2D3 Regulation of intracellular
calcium (Sergeev and Rhoten 1998) hCG Induct
ion of expression of apoptotic genes (Srivastav
a, et al 1998) Selenite and Generation of
oxidative free radicals selenocystamine (Stewart,
et al 1999) Some NSAIDS Inhibition of
cyclooxygenase-2 gene expression retinoids (Danne
rberg)
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Mechanisms of action of chemopreventive agents
that induce apoptosis-b
Agent Mechanism (Reference) Some NSAIDS
Increase in arachidonic acid and stimulation
of conversion of sphingomyclin to
ceramide (Chan, et al 1998) Monoterpenes Inh
ibition of isoprenylation of small G
proteins and altered gene
experssion (Gould 97) 4HPR Increase in ROS
(Delia, et al, 93 Oridate, et al 95) Resveratrol
Activation of p53 activity (Huang, 99)
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Differentiation-inducing agents-1

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Differentiation-inducing agents-2
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Retinoic Acid is Critical for the Differentiation
of Tracheobronchial Epithelial Cells
COOH
RA
Mucous differentiation
Squamous differentiation
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CHARACTERISTICS OF INTERMEDIATE BIOMARKERS FOR
CHEMOPREVENTION STUDIES

• DIFFERENTIAL AND PROGRESSIVE EXPRESSION (OR LOSS)
  IN NORMAL, PREMALIGNANT AND MALIGNANT TISSUES
• ASSOCIATION OF MARKER EXPRESSION WITH THE CAUSAL
  PATHWAY OF CARCINOGENESIS
• MODULATION OF MARKER EXPRESSION BY THE
  CHEMOPREVENTIVE AGENT USED IN CLINICAL TRIALS
• ASSOCIATION BETWEEN MODULATION BY AGENT AND
  CLINICAL RESPONSE

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Retinoid-based cancer prevention trials
hepatomaeffective skin cancer effective for
SCC not BCC cervical cancerefective breast
cancer no effect for interim analysis lung
cancer not effective Retinoid-based cancer
treatment trials kaposis sarcomatwo trials
(response rate were 38 and 46 in phase II and
44 in Phase I/II) acute promyelocytic
leukemiacomplete remission in 83 of patients
myelodysplastic syndromeeffective in patients
with low-risk disease. thyroid cancereffective
with adjuncts with other agents breast cancer
need further investigation
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Future direction novel retinoids high effect
and low toxicity combination regimens
(synergistic interactions) LG1069 SERM
(selective estrogen receptor modulators), RA
IFN
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Summary RAs have a tremendous potential in the
prevention and treatment of cancer
patients RAs are now part of the standard of
care in the treatment of APL, kaposis sarcoma,
and several premalignant conditions including
cervical dysplasia and oral premalignant
lesions obstacles, such as toxicity and drug
resistance may be avoided with some of the newer
retinoids under development novel RAs alone and
combination with others (tamoxifen and PPAR
ligands) are extremely exciting in the future