Targeting Prostate Cancer Stem CellsCancer Stem Cells in the Headlines Forbes October 09, 2012...
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Targeting Prostate Cancer Stem Cells 1 Wendy J. Huss, PhD Department of Pharmacology and Therapeutics Roswell Park Cancer Institute Buffalo NY
Transcript of Targeting Prostate Cancer Stem CellsCancer Stem Cells in the Headlines Forbes October 09, 2012...
TargetingProstateCancerStemCells
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WendyJ.Huss,PhDDepartmentofPharmacologyandTherapeutics
RoswellParkCancerInstituteBuffaloNY
Outline
• HistoricalResearch,IdentificationofCancerStemCellsinLeukemia
• TechniquestoIdentifyCancerStemCells
• Prostate(Cancer)StemCells– Identification– Therapy
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Definition of (Cancer) Stem Cells
Benign and Cancer Stem Cells: Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type. Cancer Stem Cells: Generate all heterogeneous lineages of cells within a tumor
Suggests a hierarchy of tumor initiating capabilities
Modified from Wikipedia.org
1950-1970’s G. Barry Pierce: proposed organ cell hierarchy of organogenesis of tumorigenesis
1990’s John Dick: evidence for the existence of cancer stem cells in Acute Myeloid Leukemia
Cancer Stem Cell Hypothesis is NOT New
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Cancer Stem Cells in the Headlines
Forbes October 09, 2012 Cancer Stem Cell Therapy: Real Or Just Hype? By Nathan Sadeghi-Nejad
The New Yorker September 7, 2014 The Transformation By Jerome Groopman
Fox News September 27, 2012 Common cancer treatments may create dangerous cancer stem cells By Charles Q. Choi
BBC August 1, 2012 Cancer stem cell discovery could signal 'paradigm shift' By Pallab Ghosh
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Cancerstemcell
TreatmentCancer stem cells survival
Tumor relapsed
Cancer Stem Cell Hypothesis
Modified from Collins & Maitland. Eur J Cancer (2006) 42(9):1213-8.
Self-renewal
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Cancer Stem Cell Identification
No Tumor
Disassociate Tumor
Cancer Stem Cell
Majority of Cancer Cells
Tumor Generating Assay
Tumor
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Cell Identification with Antibodies
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Flow Cytometry
Gre
en In
tens
ity
Red Intensity
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Tumor Cell Suspension Flow
Cytometry
Identify Cells
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DissociateTum
or
Labe
lCellswith
Fluo
rescen
tAntibod
y
AnalyzeCe
llswith
Flow
Cytom
etry
Lodish et. al., NCBI.
Flow Activated Cell Sorting
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Bone Marrow Transplants in Mice
Drugs Today 2002, 38(2): 103 11
www.urmc.rochester.edu/.../Craig_Jordan.htm 12
First Demonstration of Cancer Stem Cells In vivo assay for human leukemia – Demonstrated different stages of AML could engraft in irradiated mice had colony formation CD34+CD38-, but not CD34+CD38+ cells could recapitulate human AML in mice
Lapidot, et.al., Nature: 367; 645-648 (1994) 13
Common protective mechanisms between benign and cancer stem cells assays for discrimination and isolation Multidrug resistance pumps Hoechst Efflux Side Population Vybrant® DyeCycle™ Violet Side Population High Aldehyde Dehydrogenase Activity ALDEFLOUR ® Radiation Protection: ↑Chk1 & Chk2 mediated DNA repair capacity Telomerase Activity
Stem Cell Protection Mechanisms
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Hematopoietic Side Population Phenotype
Goodell et al. (1996) J. Exp Med 183: 1797-806
+ verapamil
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ABC Transporters
• ATP binding cassette transporters- the largest family of drug transporters
• Evolutionarily conserved
• Present in plasma membrane and membranes of intracellular compartments
• Use of cellular ATP to drive transport
• 7 subfamilies and 50 ABC transporters
Dean, M. and R. Allikmets (2001). J Bioenerg Biomembr 33(6): 475-479.16
ABCTransporterSuperFamilySubfamilies Moststudiedmember Roleofthemoststudiedmember
ABCA1-12 ABCA1and2 Cholesterolefflux(A1),Drugresistance(A2)
ABCB1-11 ABCB1,6,11 Multidrugresistance,Hoechst(B1)Irontransport(B6)
Bilesalttransport(B11)
ABCC1-13 ABCC1-5 Drugresistance(C1,3)Nucleotidetransport(C4,5)Chloridechannels(C7)
ABCD1-4 - -
ABCF1-3 - -
ABCE1 - -
ABCG1-8 ABCG1,2,5,8 Cholesterolefflux(G1)Toxins,drugs,dyese.g.Hoechst,rhodamine,
DCV,steroids(G2)
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Add Antibody to Recognize Cancer Stem Cells (red) and Cancer Cells (blue)
Identification of Cancer Stem Cells in Solid Tumors
18Shackleton M et al., Cell 2009;138(5):822-829
Shackleton M et al., Cell 2009;138(5):822-829
Identification of Cancer Stem Cells in Solid Tumors
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ProstateCancerisSecondCauseofCancerRelatedDeathinAmericanMen
AmericanCancerSociety
ClinicalSignificance:CastrationResistantProstateCancer
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Normaladultstemcell
Organ Stem Cells
Normal Prostate
Bladder
Urethra
Cancerstemcell
Prostate Cancer
Bladder
Cancer Stem Cells
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Schematic Depiction of the Prostatic Duct
Abate-Shen,C.andM.M.Shen(2000).GenesDev14(19):2410-2434.
Basalcellmarkers:p63,CK5,CK14
Luminalcellmarkers:AR,CK8,CK18
Non-epithelialcells(Lineagemarkers):
Endothelialcells:CD31Fibroblasts:CD45andCD34
Erythroidcells:Ter119
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StemCell?
Abcg2+ Cells are Located in Stem Cell Compartment
ABCG2-BlueAR-Red
ABCG2-Bluep63-Red
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Huss,W.J.,etal.(2005)."CancerRes65(15):6640-6650.
ABCG2 Inhibition Increases Intracellular Androgen and Androgen Receptor
Huss,W.J.,etal.(2005)."CancerRes65(15):6640-6650.
Black-HoechstGrey-DHT
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Abcg2
AR
AR
ARNovFTCKO143
Cell with a functional ABCG2 transporter
Cell with a non-functional ABCG2
transporter
AR
Differentiated cell
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Androgen Receptor (AR) Regulation by Androgens
DHT
Prostate Stem Cell Niche Tumor Stem Cell
Basal
NE
Stroma
Transit-Amplifying Cell
Stro
ma
Sig
nalin
g
Diff
eren
tiatio
n
Prostate Benign and Cancer Stem Cells
ABC transporter
Secretory Adenocarcinoma
DHT
DHT
DHT
DHT
DHT
DHT
DHT
DHT27
Prostate Stem Cell Niche Tumor Stem Cell
Basal NE
Stroma
Androgen Deprivation Therapy Recurrent Prostate Cancer
Recurrent Prostate Cancer
Diff
eren
tiatio
n
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Secretory
Adenocarcinoma
DHT
DHT
DHT
DHTDHT
DHT
Prostate Stem Cell Niche Tumor Stem Cell
Basal NE
Transit-Amplifying Cell
Stroma
Diff
eren
tiatio
nInhibition of ABC Transporters Before Androgen
Deprivation to Target Cancer Stem Cells
ABC transporter
DHT
DHTDHT DHT
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DHT
Basal NE
Stroma
Diff
eren
tiatio
nInhibition of ABC Transporters Before Androgen
Deprivation to Target Cancer Stem Cells
Secretory
Adenocarcinoma
DHT
DHT
DHT
DHTDHT
DHT
Transit-Amplifying Cell
Prostate Stem Cell Niche Tumor Stem Cell
ABC transporterDHTDHT DHT
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Hypothesis:ABCG2effluxofandrogeninhibitsprostatestemcelldifferentiationtomaintainstemcellproperties
Specificaims:1.Determinethemechanismofandrogeneffluxtomaintainstemcellproperties.ABCG2mediatedandrogeneffluxinhibitsARinducedprostatestemcelldifferentiation.2.Identifyregulatorsofthesidepopulationphenotypethatcontributetomaintainingprostatestemcellproperties.ProstatestemcellswithinthesidepopulationrequireABCG2expression.3.DeterminetheeffectofabrogatedABCG2functionontheprostatestemcellniche.ABCG2inhibitiondepletesstemcellcompartmentandtheprostateisunabletoseriallyregenerate.
Control DHT+KO143DHT KO143
DAPI
AR
Merge
ABCG2-(+DHT)
ABCG2expressingHPr-1-ARcells(SimilarResultsinCWR-R1)
NehaSabnis
Aim1:InhibitingABCG2MediatedAndrogenEffluxIncreasesARNuclearTranslocation
ABCG2-expressingHPr-1-ARcells(SimilarResultsinCWR-R1)
Aim1:InhibitingABCG2MediatedAndrogenEffluxIncreasesExpressionofLuminalDifferentiationMarkers
CK8
Actin
PSA
CK18
SOX2
NehaSabnis
DHTKo143Enzalutamide
---
+--
++-
+++
--+
-+-
Summary Aim 1 and Future Directions
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ABCG2inhibitionincreasesnuclearAR&expressionofARregulateddifferentiationmarkers
DetermineifdifferentiationisregulatedbyARactivation inhibitARanddeterminedifferentiationcapabilities
Aims• DetermineARFunctionwithABCG2Inhibition• DetermineStemCellPropertiesofABCG2ExpressingCells• DeterminetheRoleofABCG2inStemCellMaintenance
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Hypothesis- Inhibition of ABCG2-Mediated Androgen Efflux Eliminates the Prostate Cancer Stem Cell Compartment
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Non-Tumor
Tumor
TRAMP
ABCG2 Expression in Side Population from Human Prostate Specimens
Mathew et al., 2009 Cell Cycle 8:1053-61
Testing Prostate Stem Cell Properties with Tissue Recombination
Embryonic Rodent Urogenital Tract
(UGS)
(UGM)(UGE)
B. Foster
Graft in vivo under renal capsule
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Cells + collagen = no growth
Cells + UGM = growth
Tissue Recombination of rUGM and Putative Stem Cells
Rat Urogenital Sinus
Trypsinize for 60 minutes at 37oC
Rat Urogenital MesenchymeUrogenital Epithelium
FACS isolated Epithelial cellsFACS isolated Epithelial cells attached
to Rat Urogenital Mesenchyme
Graft under kidney capsule of SCID mouse
Harvest grafts after 8 weeks and analyze histologically
Rat Urogenital Sinus
Trypsinize for 60 minutes at 37oC
Rat Urogenital MesenchymeUrogenital Epithelium
FACS isolated Epithelial cellsFACS isolated Epithelial cells attached
to Rat Urogenital Mesenchyme
Graft under kidney capsule of SCID mouse
Harvest grafts after 8 weeks and analyze histologically
Side Population Assay
1.2%
Side population cells isolated from radical prostatectomy specimens
H&E analysis of side population cells + UGM
FISH analysis for epithelial species identification, IHC analysis for differentiation markers
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Serial Tissue Recombinations
Foster et al., 2013 PLoS ONE 8(1): e55062
Summary Aim 2 and Future Directions
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SidePopulationAssayenrichesforProstateStemCells
Next-DetermineifSidePopulationAssayenrichesforProstateCancerStemCells
Aims• DetermineARFunctionwithABCG2Inhibition• DetermineStemCellPropertiesofABCG2ExpressingCells• DeterminetheRoleofABCG2inStemCellMaintenance
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Hypothesis- Inhibition of ABCG2-Mediated Androgen Efflux Eliminates the Prostate Cancer Stem Cell Compartment
ABCG2 Inhibition and Androgen Retention Leads to Delayed Growth Response
42Felixunpublisheddata
Time
Sphere Formation Assay
Sabnis unpublished data 43
SamantMD,JacksonCM,FelixCL,JonesAJ,GoodrichDW,FosterBA,andHussWJ.StemCellsandDevelopment2015,24(10):1236-1251.
GangavarapuKJ,AzabdaftariG,MorrisonCD,MillerA,FosterBA,HussWJ.StemCellResearchandTherapy2013;4:132.
InhibitingABCG2FunctionReducesSphereFormingCapabilities
Primarymouseventralprostatecells
CWR-R1cells
Ko143inhibitsABCG2>ABCB1>ABCC1
The Role of Androgens in Prostate
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Apoptosisintheluminalcellcompartment
Tsujimura,A.,etal.(2002).JCellBiol157(7):1257-1265.
ABCG2nullmousemodel
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• Abcg2isdeletedembryonically.• Systemicdeletion.• Byreplacingexons3and4withaneomycincassetteviahomologousrecombination.
Zhou,S.,etal.(2002)."Bcrp1geneexpressionisrequiredfornormalnumbersofsidepopulationstemcellsinmice,andconfersrelativeprotectiontomitoxantroneinhematopoieticcellsinvivo."ProcNatlAcadSciUSA99(19):12339-12344.
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P<0.01P<0.001
Aim3:Abcg2nullprostatecellsweremoresensitizedtoreversantreatmentthanWTcontrols
ReversaninhibitsABCC1>ABCB1>ABCG2
Summary
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ABCG2isamarkerofprostate(cancer)stemcells
InhibitionofABCG2forcesprostate(cancer)stemcelldifferentiation
ARnucleartranslocation Elevateddifferentiationmarkers Decreasedcellgrowth Decreasedsphereformation
Future Directions
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Determineifdecreasedcancerstemcellsreducestumorrecurrence
Cancerstemcell
TreatmentCancer stem cells survival
Tumor relapsed
Self-renewal
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Cancerstemcell
Tumor Cell Treatment
No Tumor Relapse
Self-renewal
Cancer Stem Cell Treatment
Clinicalsignificance:Differentiationtherapyforprostatecancer
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AcknowledgmentsHuss Lab Kalyan Gangavarapu, PhD Neha Sabnis, MS Alumni Mugdha Samant, PhD Carina Felix Brent Feudale, MD Courtney Jackson Grinu Mathew, PhD Paula Sotomayor, PhD Cecilia Choy, MS Mame Diop
Pharmacology & Therapeutics Barbara Foster, PhD Candace Johnson, PhD MTMR Bryan Gillard Ellen Karasik, MS Michael Moser, PhD
Pathology Gissou Azabdaftari, MD
Biostatistics Austin Miller, PhD
Urologic Oncology James Mohler, MD Khurshid Guru, MD
Tissue Procurement Carl Morrison, MD Philip Ferenczy, Nancy Reska, & Liz Breese
Flow Cytometry Paul Wallace, PhD Earl Timm & Craig Jones
St. Jude Children’s Research Hospital Brian Sorrentino, MD
MD Anderson Mark Titus, PhD Funding NIDDK Sklarow Foundation NYSTEM
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