1. Cancer Biology
  2. Developmental Biology and Stem Cells
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Differential requirements of androgen receptor in luminal progenitors during prostate regeneration and tumor initiation

  1. Chee Wai Chua
  2. Nusrat J Epsi
  3. Eva Y Leung
  4. Shouhong Xuan
  5. Ming Lei
  6. Bo I Li
  7. Sarah K Bergren
  8. Hanina Hibshoosh
  9. Antonina Mitrofanova
  10. Michael M Shen Is a corresponding author
  1. Columbia University Medical Center, United States
  2. Rutgers, The State University of New Jersey, United States
Research Article
Cite as: eLife 2018;7:e28768 doi: 10.7554/eLife.28768
6 figures, 1 table, 6 data sets and 2 additional files

Figures

CARNs remain luminal after AR deletion.

(A) Time course for lineage-marking of CARNs and inducible AR deletion using castrated and tamoxifen-treated control Nkx3.1CreERT2/+; R26R-YFP/+ mice and Nkx3.1CreERT2/+; Arflox/Y; R26R-YFP/+ mice. (B) FACS analyses of lineage-marked YFP+ cells in total EpCAM+ epithelial cells. (C) Percentage of YFP+ cells among total epithelial cells in castrated and tamoxifen-induced Nkx3.1CreERT2/+; R26R-YFP/+ controls and Nkx3.1CreERT2/+; Arflox/Y; R26R-YFP/+ mice. Error bars represent one standard deviation; the difference between groups is not significant (p=0.51, independent t-test). (D) Expression of AR, luminal markers (CK8 and CK18), and basal markers (CK5 and p63) in lineage-marked CARNs (top) and AR-deleted CARNs (bottom). Note that all lineage-marked cells express luminal but not basal markers (arrows). Scale bars in D) correspond to 50 μm.

https://doi.org/10.7554/eLife.28768.003
Figure 1—source data 1

Quantitation of CARNs and AR-deleted CARNs in vivo.

https://doi.org/10.7554/eLife.28768.004
AR-deleted CARNs fail to generate lineage-marked cell clusters but remain bipotential during androgen-mediated regeneration.

(A) Time course for lineage-marking and androgen-mediated regeneration. (B) Percentage of single YFP+ cells or YFP+ clusters of 2 cells, 3–4 cells, and >4 cells at 4, 7, 14, and 28 days of androgen-mediated regeneration. This analysis does not include YFP+AR+ cells that fail to undergo AR deletion in the experimental mice; full quantitation of all cell populations is provided in Figure 2—source data 1. (C) YFP+ cells (arrows) in prostates of mice with lineage-marked CARNs (top) and AR-deleted CARNs (bottom) at days 4, 7, 14 and 28 days during androgen-mediated regeneration. (D) Identification of basal YFP+ cells (arrows) as progeny of CARNs (top) or AR-deleted CARNs (bottom). Scale bars in C) and D) correspond to 50 μm.

https://doi.org/10.7554/eLife.28768.005
Figure 2—source data 1

Quantitation of YFP+ cells during regeneration.

https://doi.org/10.7554/eLife.28768.006
AR-deleted CARNs and/or their progeny have defects in proliferation during regeneration and in renal grafts.

(A,B) Time course of BrdU incorporation during androgen-mediated regeneration of castrated and tamoxifen-treated control Nkx3.1CreERT2/+; R26R-YFP/+ mice and Nkx3.1CreERT2/+; Arflox/Y; R26R-YFP/+ mice. BrdU injections were performed during either days 1 through 4 (A) or days 11 through 14 (B), followed by analysis at 28 days. (C) Identification of BrdU+YFP+ cells (arrows) in control (top) and AR-deleted (bottom) prostate tissue after administration of BrdU during early stages of regeneration. (D) YFP-positive cells in control prostate tumors (top) can incorporate BrdU (arrow) but not in AR-deleted prostate tumors (bottom), after administration of BrdU during later stages of regeneration. (E,F) Percentage of BrdU+ and BrdU cells among total YFP+ cells after injection of BrdU from days 1 through 4 (E) or days 11 through 14 (F) of regeneration. Error bars represent one standard deviation; the difference in (E) is not statistically significant (p=0.34, independent t-test), but is significant in (F) (p=0.027, independent t-test). This analysis excludes YFP+AR+ cells that fail to undergo AR deletion in the experimental mice; full quantitation of all cell populations is provided in Figure 3—source data 1. (G) Schematic depiction of tissue recombination of lineage-marked CARNs with rat urogenital mesenchyme followed by renal grafting. (H) Analysis of grafts generated from lineage-marked CARNs (top) and AR-deleted CARNs (bottom); arrows in bottom panels indicate AR-expressing stromal cells surrounding the AR-negative prostate duct. Scale bars in C), D) and H) correspond to 50 μm.

https://doi.org/10.7554/eLife.28768.007
Figure 3—source data 1

Quantitation of BrdU incorporation and renal grafting data.

https://doi.org/10.7554/eLife.28768.008
Figure 4 with 1 supplement
Properties of cell lines established from CARNs and AR-deleted CARNs.

(A) Morphology and marker expression of cell lines derived from single YFP+ cells from castrated and tamoxifen-treated control Nkx3.1CreERT2/+; R26R-YFP/+ mice and Nkx3.1CreERT2/+; Arflox/Y; R26R-YFP/+ mice. The APCA lines (top) and ADCA lines (bottom) show similar bright-field morphology, expression of YFP, Foxa1, and Ki67, as well as co-expression of CK8 and CK5, but differ in expression of AR. (B) APCA and ADCA cell lines display similar cell growth at days 1, 2, 4, and 6 after plating in the absence or presence of DHT, as assessed by CellTiter-Glo assay. Results shown are from a single experiment with five technical replicates and are representative of two biological replicates after normalization with day 0 luminescent signal. (C) Colony formation by APCA and ADCA cell lines in the absence or presence of DHT. Results are from a single experiment with three technical replicates and are representative of two biological replicates. (D) Renal grafts generated from tissue recombinants of 100,000 APCA or ADCA cells with rat urogenital mesenchyme, and analyzed at 12 weeks. Bottom row shows APCA grafts treated with tamoxifen for 4 days at 7 weeks of growth to induce Ar deletion (bottom); arrows indicate cells that did not undergo Ar deletion after tamoxifen treatment. Scale bars in A) and D) correspond to 50 μm.

https://doi.org/10.7554/eLife.28768.009
Figure 4—source data 1

Epithelial cell lines established from mouse and human prostate tissue.

https://doi.org/10.7554/eLife.28768.011
Figure 4—figure supplement 1
Establishment of novel human prostate epithelial cell lines.

(A) Flow-sorting strategy to eliminate EpCAME-cadherin cells from dissociated benign human prostate epithelial cells obtained from radical prostatectomies. (B) Bright-field images of a human prostate epithelial cell line at passages 3 and 6. (C) HPE cells broadly express AR and both luminal (CK8) and basal (CK5) markers, and have more limited expression of PSA and Ki67. Scale bars in B) correspond to 100 μm, and in C) to 50 μm.

https://doi.org/10.7554/eLife.28768.010
Gene set enrichment analysis of the ADCA signature.

(A) Selected biological pathways that are enriched in the ADCA versus APCA signature. (B) GSEA plot showing enrichment in the positive tail for a signature of AR-null mouse prostate epithelial cells. (C) Cross-species GSEA showing lack of enrichment with a signature based on isolated human prostate basal and luminal epithelial populations. (D–F) Cross-species GSEA comparing the ADCA expression signature with three independent expression signatures based on tumor samples from human patients. NES: normalized enrichment score; p-value is calculated using 1000 gene permutations.

https://doi.org/10.7554/eLife.28768.012
Deletion of AR alters the ability of CARNs to serve as a cell of origin for prostate cancer.

(A) Prostate histology and marker expression in Nkx3.1CreERT2/+; Ptenflox/flox; R26R-YFP/+ (NP-CARN) and Nkx3.1CreERT2/+; Ptenflox/flox; Arflox/Y; R26R-YFP/+ (NPA-CARN) mice that have been castrated and tamoxifen-treated, followed by androgen-mediated regeneration for 1 month. Shown are representative images for hematoxylin-eosin staining (H and E) and immunofluorescence for YFP, AR, phospho-Akt (pAkt), E-cadherin (Ecad), Ki67, and cleaved caspase-3 (CC3). Arrows indicate occurrence of cell death (YFP/AR in NPA-CARN), proliferation (Ecad/Ki67), and apoptosis (Ecad/CC3). (B) Quantitation of Ki67+ and CC3+-positive cells in total Ecad+ epithelial cells in NP-CARN and NPA-CARN prostates. Error bars represent one standard deviation; differences between groups are statistically significant as determined by independent t-test. (C) Prostate tumor histology and marker expression in Nkx3.1CreERT2/+; Ptenflox/flox; KrasLSL-G12D/+; R26R-YFP/+ (NPK-CARN) and Nkx3.1CreERT2/+; Ptenflox/flox; KrasLSL-G12D/+; Arflox/Y; R26R-YFP/+ (NPKA-CARN) mice that have been castrated and tamoxifen-treated, followed by androgen-mediated regeneration for 1 month. Arrows indicate cells undergoing proliferation (Ecad/Ki67) and apoptosis (Ecad/CC3). (D) Quantitation of Ki67+ and CC3+-positive cells in total Ecad+ epithelial cells in NPK-CARN and NPKA-CARN prostates. Differences between groups are not statistically significant as determined by independent t-test (Ki67, p=0.724; CC3, p=0.507). (E) Focal neuroendocrine differentiation in NPKA-CARN tumors. Shown are H and E and immunohistochemical staining (IHC) of serial sections for Synaptophysin (Syn) and Aurora kinase A (Aurka), IHC for Foxa2 and Chromogranin A (ChrA), as well as immunofluorescence for YFP and Syn shown as an overlay and as individual channels; arrows indicate positive cells. (F) Quantitation of Syn+ cells in total epithelial cells in NPK-CARN and NPKA-CARN tumors. Scale bars for H and E and IHC in A, C,) and E) correspond to 100 μm, and in other panels to 50 μm.

https://doi.org/10.7554/eLife.28768.013
Figure 6—source data 1

Tumor phenotypes and marker quantitation.

https://doi.org/10.7554/eLife.28768.014

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain (M. musculus)NOGPMID: 15879151NOD.Cg-Prkdcscid
Il2rgtm1Sug/JicTac
Taconic (Hudson, NY)
Strain (M. musculus)Nkx3.1CreERT2PMID: 19741607Nkx3-1tm4(CreERT2)Mmsestablished by Shen lab
Strain (M. musculus)PtenfloxPMID: 11691952C;129S4-Ptentm1Hwu/JJAX #004597 (Bar Harbor, ME)
Strain (M. musculus)KrasLSL-G12DPMID: 11751630B6.129-Krastm4Tyj/NciMMHCC #01XJ6
Strain (M. musculus)ARfloxPMID: 14745012B6N.129-Artm1Verh/CnrmEMMA #02579
Strain (M. musculus)R26R-YFPPMID: 11299042B6.129 × 1-Gt(ROSA)
26Sortm1(EYFP)Cos/J
JAX #006148
Cell line (Homo sapiens)HPE-1this workAdherent cell line established from radical prostatectomy 23 tissue, sorted for EpCAM+Ecad+ cells
Cell line (H. sapiens)HPE-2this workAdherent cell line established from radical prostatectomy 23 tissue, sorted for EpCAM+Ecad+ cells
Cell line (H. sapiens)HPE-3this workAdherent cell line established from radical prostatectomy 24 tissue, sorted for EpCAM+Ecad+ cells
Cell line (H. sapiens)HPE-4this workAdherent cell line established from radical prostatectomy 25 tissue
Cell line (H. sapiens)HPE-5this workAdherent cell line established from radical prostatectomy 25 tissue, sorted for EpCAM+Ecad+ cells
Cell line (H. sapiens)HPE-6this workAdherent cell line established from radical prostatectomy 25 tissue, sorted for EpCAM+Ecad+Ngfr+ cells
Cell line (H. sapiens)HPE-7this workAdherent cell line established from radical prostatectomy 25 tissue, sorted for EpCAM+Ecad+Cd24+ cells
Cell line (H. sapiens)HPE-8this workAdherent cell line established from radical prostatectomy 26 tissue
Cell line (H. sapiens)HPE-9this workAdherent cell line established from radical prostatectomy 26 tissue, sorted for EpCAM+Ecad+ cells
Cell line (H. sapiens)HPE-10this workAdherent cell line established from radical prostatectomy 26 tissue, sorted for EpCAM+Ecad+Cd24+ cells
Cell line (H. sapiens)HPE-11this workAdherent cell line established from radical prostatectomy 26 tissue, sorted for EpCAM+Ecad+Agr2+ cells
Cell line (H. sapiens)HPE-12this workAdherent cell line established from radical prostatectomy 27 tissue
Cell line (H. sapiens)HPE-13this workAdherent cell line established from radical prostatectomy 27 tissue; sorted for EpCAM+Ecad+cells
Cell line (H. sapiens)HPE-14this workAdherent cell line established from radical prostatectomy 27 tissue, sorted for EpCAM+Ecad+Cd24+ cells
Cell line (M. musculus)ADCA-1this workAdherent cell line established from single YFP+ cell isolated from castrated and tamoxifen-treated Nkx3.1CreERT2/+; Arflox/Y; R262R-YFP/+ mouse with deletedAr (recombined) allele
Cell line (M. musculus)ADCA-2this workAdherent cell line established from single YFP+ cell isolated from castrated and tamoxifen-treated Nkx3.1CreERT2/+; Arflox/Y; R262R-YFP/+ mouse with deletedAr (recombined) allele
Cell line (M. musculus)APCA-1this workAdherent cell line established from single YFP+ cell isolated from castrated and tamoxifen-treated Nkx3.1CreERT2/+; Arflox/Y; R262R-YFP/+ mouse with intact Ar (non-recombined) allele
Cell line (M. musculus)APCA-2this workAdherent cell line established from single YFP+ cell isolated from castrated and tamoxifen-treated Nkx3.1CreERT2/+; Arflox/Y; R262R-YFP/+ mouse with intact Ar (non-recombined) allele
AntibodyAndrogen receptor (AR)Sigma (St. Louis, MO)A9853
AntibodyCytokeratin 8 (CK8)Developmental Studies Hybridoma Bank (Iowa City, IA)TROMA-1
AntibodyCytokeratin 18 (CK18)Abcam
(Cambridge, MA)
ab668
AntibodyCytokeratin 5 (CK5)Covance
(San Diego, CA)
SIG3475
AntibodyCytokeratin 5 (CK5)CovancePRB-160P
Antibodyp63Santa Cruz
(Dallas, TX)
sc-8431
AntibodyGFPAbcamab13970
AntibodyGFPRoche
(St. Louis, MO)
11814460001
AntibodyBrdUAbD Serotec MCA
(Hercules, CA)
2060
AntibodyFoxa1Abcamab55178
AntibodyKi67eBiosciences
(San Diego, CA)
14–5698, clone SolA15
AntibodyCleaved-caspase-3 (CC3)BD Pharmingen
(San Jose, CA)
559565
AntibodyProstate specific antigen (PSA)Dako
(Santa Clara, CA)
M0750, clone ER-PR8
AntibodyKrasAbcamab84573
AntibodySynaptophysin (Syn)BD Transduction
Laboratories
(San Jose, CA)
611880
AntibodyAurora A (Aurka)Abcamab13824
AntibodyChromogranin A (ChrA)Abcamab15160
AntibodyFoxa2Abnova (Taiwan)H00003170-M12
AntibodyAMACRZeta Corp
(Arcadia, CA)
Z2001
AntibodyEpCAMBioLegend118214
AntibodyE-cadherineBiosciences46-3249-82
AntibodyNerve growth factor
receptor (Ngfr)
BioLegend345108
AntibodyCd24BioLegend311008
AntibodyAnterior gradient 2 (Agr2)Abcamab1139894
AntibodyEpCAMBioLegend324208
Sequence-based reagentNkx3.1 wild-type primersPMID: 19741607DOI 10.1038/nature08361
Sequence-based reagentNkx3.1CreERT2 primersPMID: 19741607DOI 10.1038/nature08361
Sequence-based reagentCreERT2 primersPMID: 19741607DOI 10.1038/nature08361
Sequence-based reagentR262R-YFP primersPMID: 11299042
Sequence-based reagentPtenflox primersPMID: 11691952DOI: 10.1126/science.1065518
Sequence-based reagentPten wild-type primersPMID: 11691952DOI: 10.1126/science.1065518
Sequence-based reagentKrasLSL-G12D primersPMID: 11751630DOI:10.1101/gad.943001
Sequence-based reagentKras wild-type primersPMID: 11751630DOI:10.1101/gad.943001
Sequence-based reagentArflox primersPMID: 14676301DOI: 10.1084/jem.20031233
Sequence-based reagentAr wild-type primersPMID: 14676301DOI: 10.1084/jem.20031233
Sequence-based reagentArflox (recombined) primersPMID: 14676301DOI: 10.1084/jem.20031233
Sequence-based reagentArflox (not recombined) primersPMID: 14676301DOI: 10.1084/jem.20031233
Commercial assay or kitTyramide amplificationThermoFisher Scientific
(Waltham, MA)
T20922
Ccommercial assay or kitTyramide amplificationThermoFisher ScientificT30953
Commercial assay or kitTyramide amplificationThermoFisher ScientificT30954
Commercial assay or kitTyramide amplificationThermoFisher ScientificT20926
Commercial assay or kitTyramide amplificationThermoFisher ScientificT20912
Commercial assay or kitABC EliteVector Labs
(Burlingame, CA)
pk6101
Commercial assay or kitCitrate-based antigen
unmasking solution
Vector LabsH3300
Commercial assay or kitTris-based antigen
unmasking solution
Vector LabsH3301
Commercial assay or kitNovaREDVector LabsSK3800
Commercial assay or kitCellTiter-Glo 3DPromega
(Madison, Wi)
G9681
Commercial assay or kitMagMAX−96forMicroarrays
Total RNA Isolation Kit
Ambion
(Waltham, MA)
Am1839Used the ‘no spin’ protocol for RNA purification
Commercial assay or kitTruSeq Stranded mRNA
library prep kit
Illumina
(San Diego, CA)
20020595Library preparation was performed by the Columbia Genome Center using Illumina kits
Chemical compound, drugTissue Tek OCT compoundVWR Scientific
(Radnor, PA)
25608–930
Chemical compound, drugGlutamaxInvitrogen
(Waltham, MA)
35050061
Chemical compound, drugTamoxifen; TMSigmaT5648-5G
Chemical compound, drugGentamicinInvitrogen15750–060
Chemical compound, drugCollagenase/hyaluronidaseSTEMCELL Technologies
(Cambridge, MA)
07912
Chemical compound, drugModified Hank's Balanced
Salt Solution; HBSS
STEMCELL Technologies37150
Chemical compound, drugDnase ISTEMCELL Technologies07900
Chemical compound, drugY-27632 ROCK inhibitorSTEMCELL Technologies72307
Chemical compound, drug10x Earle's Balanced
Salt Solution
ThermoFisher
Scientific
14155063
Chemical compound, drugHepatocyte medium supplemented withepidermal growth factor (EGF)Corning
(Corning, NY)
355056
Chemical compound, drugMatrigelThermoFisher Scientific354234
Chemical compound, drug0.25% trypsin-EDTASTEMCELL Technologies07901
Chemical compound, drugFBSThermoFisher Scientific12676029
Chemical compound, drugDMEM/F12ThermoFisher Scientific11320033
Chemical compound, drugBrdUSigmaB5002
Chemical compound, drugDispaseSTEMCELL Technologies07913
Chemical compound, drugDihydrotestosterone; DHTSigmaA8380
Software, algorithmReal time analysis; RTAIlluminahttps://support.illumina.com/sequencing/sequencing_software/real-time_analysis_rta.htmlBase calling using this software was performed by the Columbia Genome Center
Software, algorithmbcl2fastq2IlluminaIlumina: version 2.17The sequencing data was trimmed and converted to fastq format by the Columbia Genome Center
Software, algorithmSpliced Transcripts Alignment
to a Reference (STAR)
PMID: 23104886Github: version 2.5.2bSequencing reads mapping to mouse genome (USCS/mm10) was performed by the Columbia Genome Center
Software, algorithmFeatureCountsPMID: 24227677subread.sourceforge.net
version: v1.5.0-p3
Sequencing reads mapping to mouse genome (USCS/mm10) was performed by the Columbia Genome Center
Software, algorithmR-studio 0.99.902, R v3.3.0The R Foundation for
Statistical Computing,
ISBN 3-900051-07-0
v3.3.0R language for statistical computing was used for data analysis and visualization
Software, algorithmhomoloGeneNCBI
Software, algorithmGene Set Enrichment AnalysisPMID: 16199517DOI 10.1073/pnas.0506580102GSEA was used to compares differential gene expression signatures
Software, algorithmStatistical Package for the Social Sciences; SPSS, Kolmogorov-Smirnov test, Arcsine transformation, Welch t-test, Fisher's Exact TestIBM SPSS Statistics
Software, algorithmHistological grading of
mouse prostate phenotypes
PMID: 12163397DOI 10.1016/S0002-9440
(10)64228-9
OtherMini-osmotic pumpAlzet (Cupertino, CA)0000298
Other40 µm cell strainerFalcon (Corning, NY)Fisher Scientific 352340
Other96-well Primaria plateCorningFisher Scientific 353872
Other6-well Primaria plateCorningFisher Scientific 353846
Other96-well CELLSTAR plateGreiner Bio-One
(Monroe, NC)
655090
OtherLab-Tek Chamber SlideThermo Fisher Scientific154534

Data sets

The following data sets were generated
  1. 1
The following previously published data sets were used
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5

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