Dr' John E' Niederhuber

1
Frontiers in Cancer Science

• Dr. John E. Niederhuber
• Acting Director
• Listening Learning Together
• June 19, 2006

2
Cancer Isnt a Single Disease
Men720,280
Women679,510

• Heterogeneous collection
• Distinct cancer arising from unique tissues

Prostate 33 Lung bronchus 13 Colon
rectum 10 Urinary bladder 6 Melanoma of
skin 5 Non-Hodgkins 4
lymphoma Kidney 3 Oral
cavity 3 Leukemia 3 Pancreas 2 All Other
Sites 18
Breast 31 Lung bronchus 12 Colon
rectum 11 Uterine corpus 6 Non-Hodgkins
4 lymphoma Melanoma of skin
4 Thyroid 3 Ovary 3 Urinary bladder
2 Pancreas 2 All Other Sites 22
ACS Estimated 2006 Cancer Cases from SEER
3
Cancer is a Diseaseof the Genome
It arises from changes within the DNA of our
cells during their lifespan

• Deletions
• Amplifications
• Mutations
• Translocations
• Epigenetic changes

4
A Complex Foe The essential aberrations of cancer
Adapted from Hanahan Weinberg, Cell 10057
(2000)
5
Molecular Therapeutics Proof of Principle
Germinal Center B Cell -like (GCB) T(1418)
BCL-2 Chr.2p amplification c-rel locus
1.0
0.8
GCB
0.6
Patient 1
GCB
Type 3
Probability
0.4
Activated B cell-like (ABC) Constitutive
activation of NF-kB
0.2
ABC
0.0
0
2
4
6
8
10
Overall Survival (years)
ABC
Patient 2
Lymphochips
6
Targeted Drugs Gleevec
TYR
CML

• Altered cell adhesion
• proliferation
• Inhibition of apoptosis

Mauro MJ, Drucker BJ., The Oncologist, 20016233
7
Targeted Drugs Gleevec
X

• Altered cell adhesion
• proliferation
• Inhibition of apoptosis

CML
Mauro MJ, Drucker BJ., The Oncologist, 20016233
8
Resistance Mechanisms in Targeted Drugs

• Imatinib resistance is secondary to mutations
  within the kinase domain of BCR-ABL. No novel
  pathways are involved.
• This knowledge allows investigators to focus the
  study of developing resistance on this molecular
  pathway
• Phase II trial of Dasatinib (BMS-354825)
• A multitargeted kinase inhibitor with higher
  affinity to the BCR-ABL kinase than imatinib
• 93 of patients with imatinib resistance showed
  hematologic response

9
The Cancer Challenge Strategy for Progress
To preempt the cancer process on its path to a
lethal phenotype. Cancer is a system disease.
The strategy will require a multi-intervention
approach.
10
Frontiers in Cancer Biology

• Tumor microenvironment
• The presence of cancer stem cells
• Vaccine therapy in prevention

11
Former View Tumors are autonomous cell masses
whose progression is driven by genetic
alterations accumulated over decades.

Invasive Cancer
In SITU Cancer
Genetically Altered Cell
Dysplasia
Hyperplasia
Colorectal Carcinoma
Sources Weinberg, Sci Am (1996) Brugge, Dept.
of Cell Biology, Harvard
12
Current View Tumors are organs composed of
many interdependent cell types that contribute to
tumor development and metastasis.
Invasive Cancer
In SITU Cancer
ECM
Fibroblasts, adipocytes
BloodVessel
Leukocytes, macrophages,etc
Source Weinberg, Sci Am (1996)
13
Tumor Promoting Influence of theMicroenvironment
(µE)
Factors produced in cells within the tumor mE can
alter many different aspects of tumor cell
behavior e.g.

• Growth factors produced in adjacent cells promote
  cell proliferation and survival
• Cytokines and chemo tactic factors produced by
  inflammatory cells and other stroma promote cell
  migration and invasion
• Proteases produced by the mE break down basement
  membrane, altering the architecture of tissue
  structures and migration/invasion of tumor cells

Proteases
Cytokines and chemo tactic factors
ECM
Growth and survival factors
14
The Angiogenic Switch and Antiangiogenic Therapy
Tumor secretion of angiogenic factors stimulates
angiogenesis
Rapid tumor growth and metastasis
Angiogenic inhibitors may reverse this
vascularization
Somatic Mutation
SmallAvascular Tumor
Carmeliet and Jain. Nature. 2000407249.
15
Dynamic MRI Monitoring of Response to Anti-VEGF
Antibody Therapy
Pre-treatment
Post Avastin chemo
Post Avastin 4 weeks
Courtesy of Peter Choyke, M.D., NCI Clinical Trial
16
Lung Cancer Drug Development The Akt/mTOR
Pathway
17
Prevention
Tobacco components activate the pathway in normal
human airway cells
Nicotine
NNK
a/b
GSK-3
a/b
GSK-3
a
tubulin
a
tubulin
-
-
Nicotine
NNK
- - -
- - -
LY294002
LY294002

- - -
- -
-

b
DH
E
a
-BTX

18
Prevention
Pathway activation correlates with progression of
tobacco carcinogen-induced lung lesions
Progression
19
Treatment
Development of Akt inhibitors
Preclinical
Phosphatidylinositol ether lipid analogues (PIAs)
Clinical
Early phase clinical protocols Phase 0 trials-
PIAs Phase I, II trials - rapamycin
chemotherapy, off the shelf pathway
inhibitors
20
Frontiers in Cancer Biology

• Tumor microenvironment
• The presence of cancer stem cells
• Vaccine therapy in prevention

21
Cancer Stem Cells

• Hypothesis The accumulation of deleterious
  mutations (genetic alterations) required to
  establish the cancer phenotype in a given tissue
  takes place only in cells that already have the
  capacity for self-renewal.
• CaSCs may represent novel therapeutic targets for
  treating epithelial carcinoma.

22
Tissue Stem Cell DivisionSymmetric Asymmetric
Symmetric
Stem cell
Terminally differentiated cells of mature tissue
Asymmetric
Progenitor cells
23
Normal Stem Cell
Cancer Stem Cell
Normal stem cell
Embryonic precursor
Self-renewal
Mutagenesis
Restricted progenitor
Normal fetal stem cell
Premalignant stem cell
Self-renewal
Normal adult stem cell
Malignant cancer stem cell
Self-renewal
Differentiation
Restricted Differentiation
Mature Cells (limited proliferative potential)
Benign Cancer (limited proliferative
potential )
24
Cancer Stem Cells
Tumor stem cell
Chemotherapy Radiation
Tumor
Remission
25
Stem (SP) Cells in Cell Lines

• Percent of viable
• Cell line Tumor cells with SP
  phenotype
• C6 Rat Glioma 0.4
• HS 683 Human Glioma 20
• B104 Rat Neuroblastoma 0.4
• MCF7 Human Breast 2.0
• HeLa Human Cervical Carcinoma 1.2
• SK-BR-3 Human Breast adenocarcinoma 2.0
• SK-OV-3 Human Ovarian adenocarcinoma 12
• NCI-H146 Human Small cell lung 8.0
• RD-ES Human Ewing sarcoma 7.0
• U-2OS Human Osteosarcoma 0
• SaOS-2 Human Osteosarcoma 0
• A204 Human Rhabdomyosarcoma 0

Hirschmann-Jax, PNAS 10114228 (2004)
26
Breast Cancer Stem Cells

• 2000 500 200 100
• Stem-like cells (1)
• CD44 CD24- ESA 10/10 4/4 4/4 1/6
• Non stem cells (99)
• CD44 CD24- ESA- 0/10 0/4 0/4 0/6

Number of cells injected into mammary fat pads
in NOD/SCID mice. The stem cell-like cells out
of a breast tumor are tumorigenic the rest of
the cells are not.
Source Dontu, Genes Dev 171253 (2003)
27
Self-renewal and Differentiation of Cells Grown
as Neurospheres

Uchida, Nobuko et al. (2000) Proc.
Natl. Acad. Sci. USA 97, 14720
28
Neural Stem Cells

• 34 NOD SCID mice were injected with human
  medulloblastoma or gliomablastoma multiforme
  cells grown as neurospheres, then selected for CD
  133 surface marker.
• Of 19 injected with CD 133 cells 16 (84)
  developed tumors.
• None of the 15 mice injected with CD 133- cells
  formed tumors

( of cells injected varied, with max at 50K
for medulloblastoma and 100K for glioblastoma
multiforme. All CD 133 cells were injected at
maximum .)
Singh, Nature 432 396 - 401 (2004)
29
Neural Stem Cells
Mouse Forebrain After CD 133 injection
Tumor histology low power
Forebrain injected with CD 133- tumor cells
Tumor histology high power
Singh, Nature 432 396 - 401 (2004)
30
Cancer Stem Cells

• Have ability to travel to other tissues do not
  need to acquire this characteristic
• Drug resistance not acquired but present
• High levels of ABC (ATP Binding Cassette)
  transporters actively efflux drugs (ABCG2, ABCB1,
  ABCD1)
• Molecular pathways uniquely present in HSCs and
  CSCs (Notch, Hedgehog Hh, Wnt, Bmi-1)

31
Stem Cell Conclusions

• Many solid tumors appear to have a population of
  stem cells partially resistant to chemotherapy.
• Isolating and studying these cells may give us
  new insights into cancer and therapies.
• The HH/PTCH pathway is activated in many cancers.
• Cyclopamine may be an effective drug to treat
  these tumors.
• Other options for targeting cancer stem cell.

32
Frontiers in Cancer Biology

• Tumor microenvironment
• The presence of cancer stem cells
• Vaccine therapy in prevention

33
Cervical Cancer

• Incidence
• 9,710 cases estimated in U.S. in 2006
• 470,000 cases per year worldwide
• Deaths
• 3,700 estimated deaths in U.S. in 2006
• 233,000 deaths per year worldwide

34
Human Papillomavirus (HPV) Infection and Cervical
Cancer

• Cervical cancer is the 2nd most common
  malignancy-related cause of death in women
  worldwide (Parkin et al, Eur J Cancer 2000)
• Oncogenic human papillomaviruses are the
  etiological agent for virtually all cervical
  cancers (worldwide prevalence in cervical tumors
  of 99.7)
• Majority of cervical HPV-infections are transient
  non-neoplastic productive viral infections which
  disappear within a year only approx. 1 progress
  to high grade dysplasia and cancer

35
What causes progression from HPV infection to
cervical cancer?
It is generally accepted that persistence of high
risk HPV infection is an imperative state in the
development of cervical neoplasia
The crucial factors determining persistence of
infection and its correlation with an increased
risk for cervical cancer in women with normal
immune status are still largely unknown.
Persistent infection may only occur upon targeted
infection of specific cervical cells, possibly
with stem cell properties
36
HPV VLP Vaccines in Phase III Trials
GlaxoSmithKline HPV16
HPV18
ASO4 Adjuvant (MPL Alum)
Made in insect cells Merck
HPV16 HPV18 HPV6 HPV11
Alum Adjuvant Made in yeast
70 of Cervical Ca
70 of Cervical Ca

90 of Genital Warts
IM Injections at 0, 1 or 2, and 6 months
37
Merck Phase 3 Tetravalent VaccineInterim
Analysis (Unpublished)
ATP analysis. HPV6,11,16, 18 Associated Disease
Only
ATP received 3 doses of vaccine HPV sero(-) at
day 1 and HPV DNA(-) from day 1 to month 7
cases counted starting after month 7. Average
Duration of Follow-up 1.5 Years After the Last
Vaccination
38
Will the HPV Vaccine Reach the Women Who Need it
Most?
More
developed
Less
developed
countries
countries
Breast
Pap smear
Cervix
Ovary
Endometrium
Colon/rectum
Lung
Stomach
2
00
4
00
6
00
0
2
00
4
00
6
00
Annual number of cases (thousands)
Adapted from Parkin et al, Eur J Cancer 37S4,
2001
39
Vaccine Policy, United States

• Greatest benefit if given to 10-13 year old girls
  or older girls/women who have not become sexually
  active
• Make available to poorer women
• Vaccinated women must continue to follow standard
  guidelines for cervical cancer screening
• Vaccination before becoming sexually active and
  screening after becoming sexually active could
  reduce cervical cancer by more than 90

40
Vaccine Policy, Developing World

• 80 of cervical cancer cases occur in the
  developing world
• Currently lacks high-quality screening programs
• Screening is useful for current generation
• Vaccination, because it should be given to
  adolescents, is useful for the next generation
• Reduction in cost of vaccine or organized
  distribution program will be needed

41
Predictive Medicine

• Do each individuals complete genome and proteome
• ? Assess for tumor rejection antigen(s)
• Multi parameter diagnostics to visualize patient
  in present predicting future
• Selecting drugs to redesign the behavior of
  biologic systems

42
How Do We Get There?
Put more science into clinical trials
Investigational drug
Responders
Non-responders
Pharmaco dynamic measurements
Molecular diagnostics candidate approach
Molecular diagnostics unbiased approach
Modified from American Association for Cancer
Research
43
Individualized Medicine
The hope for the future
Breast cancer
Treatment A
Molecular diagnostics
Treatment B
Prostate cancer
Treatment C
Lung cancer
Standard TX
Modified from American Association for Cancer
Research
44
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45
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46
Prevention
An FDA-approved mTOR inhibitor, rapamycin,
decreases number and size of tobacco
carcinogen-induced lung lesions
Size
Number
47
Prevention

• Impact
• Conceptual shift in tobacco-related
  carcinogenesis
• Pathway activation as biomarker in prevention
  trials
• Provides strong rationale to test mTOR inhibitors
  for lung cancer prevention

48
Approved Targeted Drugs

• Alemtuzumab (Campath)
• Arcitumomab
• Bevacizumab (Avastin)
• Bortezomib (Velcade)
• Capromab pendetide
• Cetuximab (Erbitux)
• Gefitinib (Iressa)

• Gemtuzumab (Myelotarg)
• Ibritumomab tiuxetan (Zevalin)
• Imatinib (Gleevec)
• Rituximab (Rituxan)
• Tositumomab (Bexxar)
• Trastuzumab (Herceptin)

49
Drug Discovery Directed to Tumor Stem Cells

• Proposed Study
• COMPARE Analysis with tumor stem cell marker
• e.g. CD44 and CD24
• Current Investigation
• Cloning efficiency in NCI 60 cell lines as an
  indirect measure of stemness
• Using COMPARE Analysis have identified compounds
  with highest positive and negatively correlated
  signatures.
• The positively correlated compounds have
  mechanisms of action similar to anticancer drugs
  already in use.

50
Candidate Cervical Stem Cells Show an Increased
Capacity for Binding of Papillomavirus-like
Particles (VLPs)
neg. control
VLP pos.
VLP neg
VLP neg
VLP low
VLP low
FSC
P5
FSC
VLP high
VLP high
VLP-binding
VLP neg
VLP low
VLP high
all cells
P3
Transferrin-R
P2
P1
Integrin a6

• Putative stem cell population
• high VLP binding capacity
• provides evidence for an
• increased number of
• HPV-binding sites which
• may facilitate infection

VLP neg
VLP low
VLP high
51
Summary of HPV VLP VaccineEfficacy Data in Women

• Vaccine well tolerated no vaccine-related SAEs
• gt99 seroconversion
• 95-100 protection from persistent infection by
  the types in the vaccine
• 100 protection from cervical pre-cancer by the
  types in the vaccine (up to 4 yrs post
  vaccination)
• 100 protection against external genital warts
• Limited or no protection against types not in the
  vaccine
• No evidence for inducing regression of
  preexisting lesions

52
ABC Transporters Expressed in Stem Cells
ABCB1/MDR1
ABCC1/MRP1
ABCG2
53
Absence of PTCH Leads to Unregulated SMO
x
PTCH
SMO
COS2
FU
SUFU
ci (GLI)
Nucleus
54
Preliminary Screening ResultsChemBridge Library
80 current threshold for secondary screens and
further investigation
50 threshold for a hit in study
FTC is a known specific inhibitor of ABCG2
Source Molecular Targets Development Program, NCI
55
Closer to the Goal

• NCIs HIV and AIDS Malignancy Branch reported
  IL-12 shows increased progression-free survival
  in Kaposis sarcoma patients studied at the NIH
  Clinical Center
• HPV vaccine to prevent cervical cancer is
  approved by FDA
• HER2/neu positive breast cancer patients show
  survival advantage in adjuvant setting with
  Herceptin

56
Current Prophylactic Vaccines are Based on
Purified Papillomavirus-Like Particles (VLPs)

• Empty shells composed of only the L1 major virion
  protein
• Induce high titers of virion-neutralizing serum
  antibodies after IM injection
• Non-infectious and non-oncogenic
• Vaccination with VLPs of animal PVs induces
  type-specific protection from experimental
  infection with high dose virus
• Protection passively transferred in serum
• No regression of established lesions
• No sexual transmission model

Kirnbauer et al. PNAS 8912180-4, 1992
57
HPV Virus-Like Particle Vaccine
Endocervix
Neutralizing Antibodies
Transformation Zone
Cervical Mucus
Intramuscular
Vaccination
Ectocervix
VLPs
58
Timeline HPV Vaccine Development
(1990-1998) Case control/cervical
cancer studies
(1995-2001) Prospective HPV neoplasia studies
Initial report of efficacy
First VLPs produced

• HPV16 and HPV18 discovered
• (1982-1992) Activities of HPB
  oncogenes determined

• HPV proposed as a necessary cause of cervical
  cancer
• VLP clinical trials begin

Vaccine licensure
HPV human papillomavirus VLP virus-like
particle
59
Acknowledgments

• Niederhuber Laboratory
• Olga Aprelikova, Ph.D
• Astrid Baege, M.D.
• Christina Stuelten, M.D, Ph.D.
• Simone John, M.D.
• Kendra Mabon, B.S.

• NCI-Frederick
• Jerry Collins, Ph.D.
• Jim Doroshow, M.D.
• William Farrar, Ph.D.
• Susan Mertins, Ph.D.
• Bob Shoemaker, Ph.D.

NCI Breast Cancer Premalignancy Program
60

• Cells cultured with mitoxantrone to force
  increased expression of ABCG2
• Incubated with Ph A and 10 µM test substance
  for 18 hours
• Fluorescence measured and compared to that of
  cells incubated with 10 µM FTC Ph A for 18
  hours
• lt50 FTC response reject
• gt50 of FTC response confirmed hit
• Next step Determine dose response by above
  methods mitoxanthrone assay

Pheophorbide A is effluxed from ABCG2 expressing
cells. There is low intracellular dye
accumulation.
Ph A
In the presence of FTC or other ABCG2 inhibitor,
more dye is accumulated.
Ph A Inhibitor
61
Eupatin Mitoxantrone Assay
62
Target first step in Ca stem cell differentiation
Seek agents that block this step
63
HPV and Cervical Cancer

• 92.9 of 932 cases of invasive cervical cancer
  worldwide tested positive for HPV DNA in 1995
  IARC study

• Of the 66 HPV neg cases, the 34 histologically
  adequate samples were later reanalyzed with more
  sensitive techniques. Only 2 were in fact HPV
  negative.
• Combined results yield 99.7 of ICC cases were
  HPV positive

Walboomers et al Journal of Pathology 189 12
19 1999
64
Mechanism of Assymetric Cell Division
Dogma is DNA strands are randomly distributed to
daughter cells. In murine stem cell
division, both older Watson (W)-containing
chromosome-7 chromatids were shown to go to one
daughter cell and both older Crick
(C)-containing chromatids went to the other
daughter. In three other adult cell types they
are randomly distributed.
G2 chromatids
Chromosomes


W
C

W
C

Strand segregation is cell type regulated
(Armakolas and Klar Science 2006) Gene
Regulation and Chromosome Biology laboratory, NCI
Frederick
65
The History of the Cancer-Stem Cell Hypothesis
66
Conquering Cancer

• gt 1.3 million new cancers in 2005
• (205/100,000 population)
• gt 0.57 million deaths in 2005
• (1,500 each and every day)
• Estimated overall cost in 2002 171.6 billion
• 60.9 billion direct medical
• 15.5 billion lost worker productivity
• 95.2 billion lost productivity due to premature
  death

Cancer Facts and Figures 2005, ACS Chang S. JCO,
2217 (Sept.)
67
The Paradigm Shift
Human Genome Genomics Proteomics Immunology Mechan
isms Rational Design
Research
68
U.S. Death Trends Cancer of All Sites, 1975-2003

• Decline of -1.1 since 1994
• Decline for men is -1.6 per year since 1993
• Decline for women is -0.8 per year since 1992
• Decline continues for Prostate, Breast (F),
  Colorectal, Lung (M), and many other sites

69
Burden of Illness FY 2006 Estimates
(Blue numbers are ranks)
70
Side Population Cells
Scharenberg, Blood 99507 (2002)
71
Isolating Cancer Stem Cells ina Cell Line
C6 Cell Line Side Population
Dean, Laboratory of Genomic Diversity, NCI
72
Cancer Stem Cells

• Mimic embryonic and adult stem cells
• Long life-span, relative quiescence
• Increased telomerase activity enabling mitosis
  without shortening telomeres
• Co-segregate DNA strands to preserve genetic
  information
• Active DNA-repair capacity
• Resistance to apoptosis
• Can regenerate a tumor

73
Isolating Tissue/Cancer Stem Cells

• Efflux Hoechst dye (ABC transporters)
• CD surface antigens
• Ability to grow in non-adherent conditions
• Morphology
• Sca-1, OCT4/Pou5f1
• Deficient in expression of connexins and gap
  junctional intercellular communication

74
Isolating stem cells in cell lines
Serum Serum Free
90 80 70 60 59 40 30 20 10 0
of spheres/ 10,00 cells
EGF
bFGF
EGFbFGF
No GF

• Few single cells cultivated in non-adherent
  conditions survive
• Those that do survive form floating spherical
  colonies
• Colony cells have demonstrated stem cell
  characteristics

Source Dontu, Genes Dev 171253 (2003)
75
Opportunities for Effective Therapies in Cancer
Stem Cells

• Blocking transition from stem cell to stem 1
  (proliferative cell type)
• Drug resistance not acquired but present
• High levels of ABC (ATP Binding Cassette)
  transporters actively efflux drugs (ABCG2, ABCB1,
  ABCD1)
• Molecular pathways uniquely present in HSCs and
  CSCs (Notch, Hedgehog (Hh), Wnt, Bmi-1)

76
ABCG2 as a Molecular Target
Pheophorbide A
X
X
ABCG2 Inhibitor
H460 cell line Lung Cancer
Blocked Exporter
A high throughput drug screen has been developed
in the Molecular Targets Development Program to
identify ABCG2 inhibitors. First pass screening
on over 100,000 compounds has identified several
potential inhibitors. These agents may be
effective against cancer stem cells.
77
ABCG2 Inhibitors

• GF120918 (Elacridar)
• Tariquidar
• Others to come
• ? Antibodies to ABCG2

78
Cyclopamine Treatment of Prostate Xenograft
Mice injected with human prostate cell line PC3.
Mice treated with 50 ug/kg/day cyclopamine. Gene
expression by RT-PCR.
Source Karhadkar, Nature 431707 (2004)
79
Hedgehog Signal Transduction
SHH
Extracellular
PTCH
SMO
COS2
Cytoplasm
FU
SUFU
ci (GLI)
PKA
Nucleus
80
The Lily and Cyclopamine
81
Cyclopamine Inhibits SMO
SHH
Cyclopamine
PTCH
SMO
COS2
FU
SUFU
ci (GLI)
Nucleus
82
Appearance of tumors before and after treatment
with topical cyclopamine
Source Tas Avci, Eur J Dermatol 1496 (2004)