Drug Resistance, Signaling Pathways: Roles in Cancer Stem Cells

1
Drug Resistance, Signaling Pathways Roles in
Cancer Stem Cells?

• James McCubrey
• Department of Microbiology Immunology
• Brody School of Medicine at East Carolina
  University
• Greenville, North Carolina, USA

2
Breast Cancer, the big cancer for women.

• Approximately 1 in 7 women will develop breast
  cancer.
• In the USA, gt200,000 women will be diagnosed with
  breast cancer this year, 2,000 men will get
  breast cancer.
• Unfortunately, gt40,000 women will die from breast
  cancer this year, 400 men will die from breast
  cancer.
• Median age of diagnosis-61 (women), 71 (male)
  years.
• Caused by inherited and non-inherited genetic
  mechanisms.
• Surgery and chemotherapy commonly used to treat
  breast cancer.
• Once breast cancer is metastatic, it is very
  difficult to effectively treat.

3
Problems in Breast Cancer Therapy

• Herceptin treatment only appropriate for those
  breast cancer patients which exhibit HER2
  overexpression (25).
• Surgery does not remove all breast cancer stem
  cells.
• Chemotherapy does not kill all breast cancer stem
  cells.
• End result relapse/reoccurrence of cancer,
  perhaps a drug resistant cancer more difficult to
  treat.
• Models to investigate drug resistant cancer.
  Altered drug transporter expression, altered
  signaling pathways.
• Breast Cancer Stem cells resemble drug resistant
  cells.

4
Classification of Breast Cancers

• Luminal (A B), comprise 70 of breast cancers
• Originate in the inner, or luminal cells that
  line
• mammary ducts.
• Estrogen Receptor (ER) positive, PR, Luminal A
  is HER2-.
• Luminal A-of all breast cancer best prognosis.
• Only 15 of Luminal A mutant at p53.
• Luminal B is HER2 and lymph node.
• Triple Negative-Basal, comprise 13-25.
• Arise from outer, basal cells that line the
  mammary
• ducts, express cytokeratins 5 6.
• ER-, PR-, HER2-, mutant at p53, maybe HER1.
• Poor prognosis, frequent relapse and distant
  metastasis.

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Classification of Breast Cancers (Cont.)

• HER2, comprise 7-12
• ER-, PR-, HER2, mutant at p53.
• HER2 breast cancer can be treated with
  Herceptin.
• Unclassified/Normal Breast-like, comprise 6-10.
• Small breast cancers, in general good prognosis.
• Racial Disparities.
• Basal more common with African Americans, more
  aggressive cancers, also diagnosed earlier in
  life.
• Associated with lack of breast feeding and excess
  
• tummy weight.
• Less Luminal A cancers in African Americans, in
  premenopausal women.

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Breast Cancer Cell Models

• MCF-7
• Estrogen Receptor (ER) positive luminal breast
  cancer.
• WT at p53, PTEN
• Mutant at Caspase-3, PIK3CA
• Weakly metastatic.
• Low percentage of CD44?CD24? Cells (2-3) CIC
• T47D
• ER, luminal breast cancer.
• Mutant at p53, PIK3CA.
• Non metastatic.
• Low percentage of CD44?CD24? Cells (2-3) CIC

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Breast Cancer Cell Models

• MDA-MB-231
• ER- basal breast cancer.
• Mutant at p53, Ki-Ras, B-Raf
• WT PIK3CA, PTEN
• Metastatic
• High percentage of CD44?CD24? Cells (gt85)
  CIC.
• Mammospheres can be isolated from all
• three cell lines.
• Mammospheres enriched in Cancer Initiating Cells
  
• CIC.

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Doxorubicin (Adriamycin)

• Doxorubicin
• Antibiotic first isolated from Streptomyces
  peucetius in early 1960s.
• Mechanisms of Action
• Intercalates between adjacent nucleotides of DNA.
• Topisomerase Inhibitor.
• Metabolism of drug causes formation of free
  radicals.

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Isolation of Drug Resistant (DR) Cells.

• Goal to determine roles of signaling pathways in
  drug resistance and ability to target DR cells.
• DR cells resemble cancer initiating cells (CICs),
  also known as cancer stem cells (CSC).
• Drug resistant cells isolated by culture in
  medium containing 10 or 25 nM doxorubicin for 3
  months.

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Doxorubicin resistant lines were isolated by
selection of cells which would grow in medium
containing 25 nM doxorubicin for 6 monthno
added oncogene in these cells.
This MTT growth comparison experiment with normal
and drug resistant was performed in 10 FBS
after incubation with the different
concentrations of the drugs for 4 days.
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Doxorubicin IC50
10-25 nM
100-400 nM
5,000 nM
CD44 /CD24- Cells
2.5
6.2
MDA-MB-231/DoxS
MDA-MB-231/DoxR
MDA-MB-231/DoxR CIC
Doxorubicin IC50
800-1,000 nM
200-250 nM
gt2,000 nM
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Increased fraction of CD44?/CD24 ? Phenotype in
Doxorubicin Resistant Cells.
Increased CD44?CD24? presence is associated with
CICs in many different cell types.
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Increased Doxorubicin Resistance in Cancer
Initiating Cells.
CICs purified from DoxR cells and normally
cultured in absence of FBS in presence of EGF and
FGF in special flasks treated to prevent
adherence of cells to avoid differentiation.
This comparison experiment with normal, drug
resistant and CIC cells was performed in 10
FBS.
14
CICs are cross-resistant to many
chemotherapeutic and hormone based drugs.
Doxorubicin, paclitaxel, 5-fluoruracil are
chemotherapeutic drugs used in breast cancer
therapy.
CICs are hyper-sensitive to Berberine.
Tamoxifen and Isoliquiritigenin target ER.
Experiments performed in 0.5 FBS.
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Berberine

• Isolated from roots, stem and bark from plants
  including Coptis chinensis , Berberis
  (goldenseal). Used in traditional medicine for
  many diseases, including diabetes, lowers
  cholesterol (LDL) and triglycerides. Has been
  used to treat many human diseases for many years.
• Mechanisms of Action (Multiple Effects)
• Berberine activates AMP-activated protein kinase
  (AMPK), and ERK, and suppresses proinflammatory
  cytokines, and reduces MMP-9 expression.
• Decreases Bcl-2 family expression (Bcl-XL,
  Mcl-1)
• Decreases NF-kB expression

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Increased Akt and ERK Activation in Drug
Resistant Cells.
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NGAL is Expressed In Some Drug Resistant Cells
DoxS
10nM DoxR
25nM DoxR
Resistance Phenotype
0
MCF-7 ER, Luminal
T47D ER, Luminal
MDA-MB-231 ER-, Basal, Metastatic
NGAL forms complex with MMP-9 and is
associated with increased metastasis in many
cancer types.
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Effects of Doxorubicin on CaM-KIV, Akt and ERK
Expression in T47D Cells
20
Synergy between CaM-K Inhibitors and
Doxorubicin In Drug Resistant Cells.
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Effects of CaM-K Inhibition on ERK Activation In
Doxorubicin Sensitive and Resistant Cells.
Doxorubicin is inducing more ERK activation in
drug resistant cells.
Active CaM-K inhibitor KN93 is suppressing ERK
activation in response to doxorubicin and also in
response to FBS in drug resistant cells.
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CICs are hyper-sensitive to CaM-K Inhibitor and
Rapamycin.
Inactive CaM-K inhibitor does not show
differential effects.
Rapamycin effective on CICs MEK inhibitor also
shows activity on CICs.
Experiments performed in 0.5 FBS.
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Summary Targeting Signaling Pathways in Breast
Cancer Initiating Cells (CICs).

• CICs are usually resistant to multiple
  chemotherapeutic drugs and hormonal therapy
  (tamoxifen).
• CICs more sensitive to CaM-K inhibitors than drug
  sensitive cells.
• Inactive CaM-K inhibitor does not distinguish
  between drug sensitive, drug resistant or CICs.
• CICs more sensitive to Rapamycin.
• CICs more sensitive to Berberine.

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Involvement of Signaling Pathways in
Migration/Motility

• Estimate of Invasiveness.
• Goal to Determine roles of Raf/MEK/ERK, PI3K/Akt
  and CaM-K pathways in Invasiveness.
• Raf/MEK/ERK pathway controls activity of certain
  transcription factors (e.g., Twist, Snail, Slug)
  involved in Invasiveness.
• Previous studies determined inhibiting
  Raf/MEK/ERK pathway inhibited migration in
  breast cancer.

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Inhibiting CaM-Ks or EGFR Inhibits Wound Healing
KN-92
Control
Control
KN-92
KN
-
92
Control
KN
-
92
Control
AG1478
KN-93
AG1478
KN-93
AG1378
KN
-
93
KN
-
93
AG1378
0 hours
24 hours
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Increased Wound Healing and CaM-KIV in Drug
Resistant Cells.
T24 hr
T0
MDA-MB-231/ DoxS
MDA-MB-231/ DoxS
MDA-MB-231/ DoxR
MDA-MB-231/ DoxR
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Development of a CaM-KIV Conditional System.
Increased motility with elevated CaM-KIV.
Increased CaM-KIV and ERK when docycycline
removed (Tet-off system).
MDA-MB-231Transfected withCaM-KIV
MDA-MB-231Empty Vector Controls
C
D
A
B
Increased migration with elevated CaM-KIV.
-

-

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Summary Targeting CaM-K in Drug Resistant Cells.

• CICs sensitive to CaM-K inhibitors.
• CaM-K inhibitors synergize with doxorubicin to
  induce death in drug resistant cells.
• CaM-K inhibitors represent novel approach to
  treat drug resistant breast cancer.
• What is the mechanism of synergyinhibition of
  ERK or inhibition of Akt or inhibition of other
  target?
• Are CaM-Ks involved in metastasis?

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Acknowledgments

• East Carolina University
• Linda Steelman
• Stephen Abrams
• William Chappell
• Michelle Lahair
• Richard Franklin

University of Wroclaw, Wroclaw, Poland Andrzej
Dzugaj

Jagellonian University Krakow, Poland Piotr
Laidler
Nencki Institute, Warsaw, Poland Jolanta
Baranska
National Academy of Sciences of Ukraine Kyiv,
Ukraine Lyudia Drobot.
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