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. 2013 Mar;15(3):274-83.
doi: 10.1038/ncb2697. Epub 2013 Feb 24.

Lineage Analysis of Basal Epithelial Cells Reveals Their Unexpected Plasticity and Supports a Cell-Of-Origin Model for Prostate Cancer Heterogeneity

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Free PMC article

Lineage Analysis of Basal Epithelial Cells Reveals Their Unexpected Plasticity and Supports a Cell-Of-Origin Model for Prostate Cancer Heterogeneity

Zhu A Wang et al. Nat Cell Biol. .
Free PMC article

Abstract

A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumour subtypes that differ in their prognosis and/or treatment response. We now show that initiation of prostate tumours in basal or luminal epithelial cells in mouse models results in tumours with distinct molecular signatures that are predictive of human patient outcomes. Furthermore, our analysis of untransformed basal cells reveals an unexpected assay dependence of their stem cell properties in sphere formation and transplantation assays versus genetic lineage tracing during prostate regeneration and adult tissue homeostasis. Although oncogenic transformation of basal cells gives rise to tumours with luminal phenotypes, cross-species bioinformatic analyses indicate that tumours of luminal origin are more aggressive than tumours of basal origin, and identify a molecular signature associated with patient outcome. Our results reveal the inherent plasticity of basal cells, and support a model in which different cells of origin generate distinct molecular subtypes of prostate cancer.

Figures

Figure 1
Figure 1
High frequency of prostate basal stem/progenitor cells in sphere formation and tissue reconstitution assays. For all analyses, tamoxifen-induced CK5-CreERT2; R26R-YFP/+ mice were analyzed at 14 days after tamoxifen treatment. (a) Immunofluorescence staining showing co-localization of YFP with CK5 in basal cells (arrowheads) of the anterior prostate. (b) Purification of YFP+ basal cells from dissociated prostate tissue by flow cytometry. (c) Flow-sorting of YFP+ cells shows that 98.7% are CD49f+, and 8.0% are LinSca-1+CD49fhi cells. (d,e) Flow sorting of LinSca-1+CD49fhi cells (panel d, 0.6% of total Lin cells) shows that 24.4% are YFP+ (e). (f) Quantitation of sphere formation from 80,000 or 20,000 dissociated prostate cells, showing the number of total spheres as well as YFP+ spheres. Each experiment was performed six times, using two replicates each from three independent mice; error bars correspond to standard deviation and show variability between the six samples. Inset shows epifluorescence detection of YFP expression in spheres (arrow). (g) Quantitation of sphere formation from 3,000 dissociated YFP+ cells isolated from CK5-CreERT2; R26R-YFP/+ mice at 2 months or 12 months of age. The frequencies at these two stages are not statistically different by two sample t-test. (h) Serial dilution analysis of purified YFP+ cells in assays of prostate duct formation in renal grafts. (i) Extreme limiting dilution analysis of data in (h). (j) YFP fluorescence of a renal graft attached to a portion of kidney tissue. (k,l) Hematoxylin-eosin staining of a tissue section from a renal graft generated from purified YFP+ basal cells; luminal (lum) and basal (bas) cells are indicated (l). (m-o) Analysis of YFP together with CK5 expression in basal cells (arrowheads, m), CK18 in luminal cells (arrows, n), and AR (o) in renal grafts. Scale bars in a,m-o correspond to 50 microns, in k,l to 100 microns, and in j to 1 mm.
Figure 2
Figure 2
Detection of rare bipotential basal progenitors during prostate regeneration in vivo. (a) Lineage-tracing strategy during prostate regeneration of CK5-CreERT2; R26R-YFP/+ mice. (b,c) Co-localization of YFP with CK5-expressing basal cells (arrowheads) in regenerated CK5-CreERT2; R26R-YFP/+ anterior prostate (b) and dorsolateral prostate (c). (d-f) Most YFP+ cells (arrowheads) do not express the luminal marker CK18, although rare YFP+CK18+ (arrow, e) and YFP+AR+ (arrow, f) luminal cells can be detected after one round of regeneration. (g) Ki67 immunostaining at two days after androgen administration shows that most luminal and some basal cells (arrowhead) undergo proliferation. (h) Quantitation of cell proliferation assays during regeneration, showing that 7.4% of basal cells (n=510/6,929) and 88.7% of luminal cells (n=6,708/7,565) were BrdU+ after 12 days of incorporation; 8.0% of basal cells (n=426/5,326) and 90.1% of luminal cells (n=6,945/7,709) were Ki67+ at 2 days of regeneration; and 8.1% of basal cells (n=380/4,700) and 85.1% of luminal cells (n=7,094/8,333) were Ki67+ at 4 days of regeneration; 3 animals were analyzed for each experiment. See Supplementary Fig. S3 and Supplementary Table S1 for additional data. (i) Lineage-tracing strategy during serial regression and regeneration. (j-l,n) YFP+ luminal cells (arrows) that co-express AR (k), Nkx3.1 (l), and CK18 (n) are more frequently observed after three rounds (j-l) and five rounds (n) of serial regeneration. (m) Castration-resistant luminal cells can be detected after four rounds of regression. (o) The frequency of luminal cells among total YFP+ cells during regeneration in anterior prostate is 0.04% (n=5/11,427, 3 animals) after 1 round, 0.07% (n=13/18,025, 5 animals) after 1 round using an alternate protocol, 0.03% (n=3/10,249, 3 animals) after 1 round in 12-month old mice, 0.6% (n=56/9,129, 3 animals) after 3 rounds, and 3.4% (n=303/8,875, 3 animals) after 5 rounds. The 1 round frequencies are not statistically different, while p<0.0001 for frequencies of different rounds by χ2 test. See Supplementary Fig. S4 and Supplementary Table S1 for additional data. Abbreviations: AP, anterior prostate; DLP, dorsolateral prostate. Scale bars in b-g,j-n correspond to 50 microns; error bars in h,o correspond to standard deviation and show variability between animals.
Figure 3
Figure 3
Detection of rare bipotential basal progenitors during prostate homeostasis. (a) Time course of lineage-tracing analysis in hormonally intact CK5-CreERT2; R26R-YFP/+ mice. (b-d) Co-localization of YFP and CK5 in prostate basal cells in mice at 4 months (b), 6 months (c), and 12 months (d) of age. (e) Detection of YFP+CK18+ luminal cells (arrow) at 12 months of age. (f) The frequency of luminal cells among total YFP+ cells during homeostasis is 0.02% (n=2/8,848) at 4 months, 0.5% (n=57/10,572) at 6 months, and 3.0% (n=227/7,638) at 12 months; 3 animals were analyzed at each time point. p<0.0001 for frequencies at different time points by χ2 test. (g) Graphical summary of BrdU incorporation analyses during homeostasis; 3 animals were analyzed for each experiment. BrdU incorporation frequencies at different time points are not statistically different by χ2 test. See Supplementary Table S1 for additional data. (h) Strategy for analyses of cell proliferation at three different ages in wild-type C57BL/6 mice. At 2 months, 6 months, or 12 months of age, BrdU was administered for 12 days followed by analysis. (i-n) Analysis of co-localization of BrdU immunostaining with CK5 (i,k,m) or CK18 (j,l,n); arrowheads indicate BrdU-positive basal cells, and arrows indicate BrdU-positive luminal cells. Scale bars in b-e,i-n correspond to 50 microns; error bars in f,g correspond to standard deviation and show variability between animals.
Figure 4
Figure 4
Basal cells are a cell type of origin for prostate tumors. (a) Time course for tumor formation in hormonally-intact CK5-CreERT2; Ptenflox/flox; R26R-YFP/+ mice. (b-d) Hematoxylin-eosin staining of anterior prostates showing slight epithelial hyperplasia at 1 month after induction (b), low-grade PIN at 3 months after induction (c), and high-grade PIN at 6 months after induction (d). (e) High-grade PIN in Nkx3.1CreERT2/+; Ptenflox/flox; R26R-YFP/+ anterior prostate at 3 months after induction. (f-l) Marker analysis of PIN lesions in CK5-CreERT2; Ptenflox/flox; R26R-YFP/+ anterior prostate. (f) Phosphorylated Akt (pAkt) can be detected in basal cells at 1 month after induction. (g,h) Ki67 immunoreactivity can be detected in basal cells at one month after induction, prior to PIN lesion formation (g), as well as at three months after induction (h). (i,j,k) Most transformed cells at three months after induction do not express CK5 (i), but instead express CK18 (j) and AR (k). (l) CK5+CK18+ intermediate cells (arrowhead) can be detected in PIN lesions at three months after induction. (m) Quantitation of basal (CK5+CK18), luminal (CK5CK18+), and intermediate cells (CK5+CK18+) in YFP+ prostate cells of CK5-CreERT2; Ptenflox/flox; R26R-YFP/+ and Nkx3.1CreERT2/+; Ptenflox/flox; R26R-YFP/+ mice at the indicated times after induction. Scale bars in b-e correspond to 100 microns, and in f-l to 50 microns.
Figure 5
Figure 5
A luminal origin gene signature that is prognostic for human prostate cancer outcome. (a) Scatter-plot of the two main components from a Principal Components Analysis based on 14,063 genes, capturing 55% (dimension 1) and 21% (dimension 2) of the data variability. (b) Scatter-plot without the control samples. Abbreviations: Bc, basal origin control; B3, basal origin 3 months post-induction; B6, basal origin 6 months post-induction; Lc, luminal origin control; L1, luminal origin 1 month post-induction; L3, luminal origin 3 months post-induction. (c) GSEA comparison of basal origin “initiation” signature (B3 relative to Bc) to luminal “initiation” signature (L1 relative to Lc) shows strong enrichment in both directions. (d) GSEA comparison of basal origin “progression” signature (B6 relative to B3) to luminal “progression” signature (L3 relative to L1) also shows strong enrichment in both directions. (e,f) GSEA shows that genes up-regulated in the L3 versus B6 gene signature are strongly enriched in a human signature corresponding to lethality due to prostate cancer (e), but not the converse (f). (g) GSEA shows biological pathways significantly enriched in the L3 versus B6 gene signature; p-value is estimated using 1,000 sample permutations. Pathways in red are up-regulated in luminal origin tumors while pathways in blue are up-regulated in basal origin tumors. (h,i) Kaplan-Meier analysis shows that the LOLES (corresponding to the 68 genes to the left of the dashed line in e) stratifies patients from two independent cohorts into groups with different rates of biochemical recurrence (red curve, “luminal-like” group, 37 patients in h, 52 patients in i; blue curve, “non-luminal-like” group, 42 patients in h, 79 patients in i). (j) Kaplan-Meier analysis shows that the LOLES stratifies patients from the Swedish “watchful waiting” cohort into a “luminal-like” group (red curve, 132 patients) and a “non-luminal-like” group (blue curve, 131 patients) with different survival outcomes (82 months versus 123 months at 50% survival). (k) C-statistics analysis shows that the LOLES improves the prognostic value of Gleason score in the Swedish cohort from 0.76 to 0.82, with the 95% confidence intervals and p-values shown.
Figure 6
Figure 6
Two models for prostate epithelial lineage relationships and cell of origin for cancer. (a) In a conventional lineage hierarchy model, luminal and basal lineages are independently maintained by largely unipotent stem/progenitor cells in the normal adult prostate epithelium. However, luminal and basal progenitors can generate the other cell type during prostate regeneration and tissue homeostasis (dashed lines); in the case of luminal stem/progenitor cells, it remains unclear whether such bipotentiality (blue dashed line) is displayed only by CARNs in the regressed state. In the case of the basal lineage, bipotential stem cells are relatively rare (approximately 0.05%), while basal cells that can display stem cell properties in sphere formation and tissue reconstitution assays are more common (approximately 4%), and perhaps might correspond to transit-amplifying cells. Oncogenic transformation of either luminal or basal cells by inactivation of Pten results in tumors with histologically similar luminal phenotypes, but tumors arising from basal cells first undergo basal cell proliferation and subsequently luminal differentiation. Tumors may arise from stem cells (dark red jagged arrows) or may also be derived from more differentiated cell types (light red jagged arrows). (b) In a stochastic progenitor model, basal cells within an intact prostate epithelium randomly display stem/progenitor properties at very low frequencies (orange), giving rise to luminal cells and being capable of self-renewal. (Luminal cells could conceivably follow a similar stochastic progenitor model, but this is not shown.) After tissue dissociation, however, the probability of such random basal stem/progenitor cells may be greatly increased. Oncogenic transformation of normal basal cells and/or stochastic basal progenitors leads to luminal differentiation and tumor formation; however, it is unlikely that stochastic progenitors represent the sole cell of origin following Pten deletion, given the rarity of these progenitors versus the frequency of observed PIN lesions.

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