targeting prostate cancer stem cells · cancer stem cell therapy: real or just hype? by nathan...

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

. .. . .. .

. .. . . . . . .

. . .

. . .

. . .. . .. . .

. . . . .

.

. . .. .

.. . .

. . .

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


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


Basal NE


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



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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targeting prostate cancer stem cells · cancer stem cell therapy: real or just hype? by nathan...

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