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. 2011 Jan 18:2:162.
doi: 10.1038/ncomms1159.

Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling

Vinagolu K Rajasekhar  1 , Lorenz Studer, William Gerald, Nicholas D Socci, Howard I Scher
Affiliations
Free PMC article

Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling

Vinagolu K Rajasekhar et al. Nat Commun. .
Free PMC article

Abstract

Androgen depletion is a key strategy for treating human prostate cancer, but the presence of hormone-independent cells escaping treatment remains a major therapeutic challenge. Here, we identify a minor subset of stem-like human prostate tumour-initiating cells (TICs) that do not express prostate cancer markers, such as androgen receptor or prostate specific antigen. These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively. The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166. Such triple-marker-positive TICs recapitulate the original parent tumour heterogeneity in serial xeno-transplantations indicating a tumour cell hierarchy in human prostate cancer development. These TICs exhibit increased nuclear factor-κB activity. These findings are important in understanding the molecular basis of human prostate cancer.

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Figures

Figure 1
Figure 1. A subset of human prostate TICs exhibits sphere-forming potential and multipotency.
Unless stated otherwise, human prostate CWR22 OT-tumours were used. (a) Phase contrast images of primary sphere formation by the total tumour cells. Scale bar, 100 μm. (b) Time course of primary sphere formation (sphere number per 8×105 total tumour cells plated, n=4). (c) Time course of primary sphere growth (average number of cells per primary sphere, n=4). (d) Efficiency of sequential derivation of primary (blue circle), secondary (green square) and tertiary spheres (black triangle) determined as a function of equal numbers of starting cells from total tumour, primary spheres and secondary spheres, respectively; n=4. (e) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) at the orthotopic (blue, tumour cells; red, primary sphere cells) and subcutaneous (black, tumour cells; green, primary sphere cells) sites. Data represents the 4-week end-point results following transplantation. Mean±s.d. (n=4). (f, g) CWR22 OT-tumour (parent tumour), the tumour cell-derived primary spheres (spheres), the sphere-derived OT-tumour (sphere tumour) and human patient primary prostate tumour (primary tumour) were used for comparative immunohistochemistry (IHC). Scale bar, 50 μm. (f) Comparative IHC for expression of differentiated prostate cell markers (AR, PSA and MET) in primary spheres versus tumours. (g) Comparative IHC for expression of markers for epithelial cells (E-cadherin) versus differentiated cells in the tumour, namely neuroendocrine cells (synaptophysin), myoepithelial cells (smooth muscle actin, SMA), and mesenchymal cells (vimentin). (h) Western blots of whole cell extracts of the parent tumour, primary spheres and sphere tumour analyzing the expression of markers for prostate basal cell (CK5), luminal cell (AR, PSA, NKX3.1, CK8 and CK18), primary prostate cancer cell (MET, αB-crystallin and maspin), putative stem cell (Musashi-1, MSH-1), basal epithelial stem cell (SOX9), cell polarity/differentiation (ZO-1) and loading control (glyceraldehyde 3-phosphate dehydrogenase, GAPDH).
Figure 2
Figure 2. Prospective isolation and characterization of human prostate TICs.
(a) Percent marker expression in parent tumours (red) or the sequentially derived primary (green) and secondary (blue) spheres. Mean±s.d. (n=10). (b) Primary sphere formation is affected by the markers expressed in tumour cells (primary spheres/5,000 tumour cells). Mean±s.d. (n=10). (c, d) Percent coexpression of TRA-1-60 and (c) human-specific-PSMA or -EpCAM or (d) mouse-specific MHC Class I H-2KD or -Sca-1 in tumour cells. Mean±s.d. (n=4). (e) Immunofluorescence labelling of TRA-1-60, CD151 and CD166 in primary spheres and 4′-6-diamidino-2-phenylindole (DAPI) represents counterstain. Scale bar, 50 μm. (f) Percent combinatorial expression of the triple-markers in tumour cells. Mean±s.d. (n=8). (g) Marker-specific primary sphere-formation efficiency (determined as above in b). Mean±s.d. (n=5).
Figure 3
Figure 3. In vivo tumour-initiation by purified human prostate TICs.
(a) Limiting dilution experiments for determining percent tumour-initiation by equal numbers of sphere cells, TRA-1-60-positive cells or triple positives transplanted at OT or SC sites. Blue, 10,000 cells; green, 1,000 cells and red, 200 cells. Data represents the 4-week end-point results following transplantation. (b–h) Unless otherwise stated, 5,000 marker-positive or total tumour (unsorted control) cells were used per transplantation. (b) Representative SC tumour growth at 5 weeks following transplantation. Unsorted represents a control tumour-derived from transplantation of 2×106 total tumour cells. (c) Tumour-initiation efficiency (represented as tumour volume) of the marker-positive cells at 5 weeks following transplantation. (d) Upper panel: Dark field images of primary spheres formed by marker-positive cells. Lower panel: hematoxylin and eosin staining of tumours derived from different marker-positive tumour cells. Scale bar, 100 μm. (e) Relative levels of G1 (yellow), G2 (blue) and S (red) phases of cell cycle in the different marker-positive tumour cells. Unsorted represents total tumour cells. (f) Percent triple-marker expression in sequentially passaged OT-tumours that were initially derived from the triple-marker-positive tumour cells. Percentage triple-marker-positive cells in the parent tumour form the control. (g) Maintenance of triple-marker-positive cells in the secondary passaged tumours derived from indicated marker-specific primary tumour cells. Unsorted is as described in (b). Orange-triple-marker-positive cells and purple-triple-marker-negative cells. (h) Immunoblot analysis of whole cell extracts of the tumours derived from marker-positive tumour cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) represents the loading control. Mean±s.d., n≥3.
Figure 4
Figure 4. Triple-marker expression in other prostate tumours and patient specimens.
(a) Comparative immunohistochemical characterization of DU-145, PC3, VCaP and PC-82 tumours. Scale bar, 50 μm. (b) Percent marker-positive cells maintained after sequential in vivo passages of DU-145 tumours that were initially derived from the triple-marker-positive cells. Blue, passage1 and light blue, passage2. Mean±s.d. (n=4). (c) In vitro primary sphere-formation and (d) In vivo tumour initiation. Prospectively isolated 2,500 marker-positive DU-145 tumour cells were used. Tumour size was determined as tumour volume. Mean±s.d. (n=4). Unsorted control represents single cells of tumour. (e) Immunofluorescence labelling of markers in fresh frozen human patient primary prostate tumour specimens, with 4′-6-diamidino-2-phenylindole (DAPI) counterstain. Scale bar, 100 μm. (f) Percent marker-positive cells in human patients' prostate tumour specimens. Mean±s.d. (n=6). (g) Percent marker-positive cells in a variety of epithelial tumour specimens from clinical patients. Red, tumour cells and green, patient-matched normal tissue cells. Mean±s.d. (n=3).
Figure 5
Figure 5. Global mRNA expression in marker-positive human prostate tumour cells.
(a) Venn-diagram of genes differentially expressed in prospectively isolated CWR22 OT-tumour cells with low (expressing EpCAM, CD44 or α2-integrin), moderate (expressing TRA-1-60, CD151 or CD166) and high (triple-marker-positives) sphere/tumour-forming efficiencies (S/TFE). Gene expression data in β4-integrin-positive (no sphere-forming) cells and the total tumour cells were used as baseline expression control. (b) Fold enrichment of gene ontology based functional categories in all the S/TFE data sets. (c–f) Catalogue of top differentially expressed genes: (c) shared among all the above three data sets, (d) shared between moderate and high S/TFE data sets, (e) expressed specifically in moderate S/TFE data set and (f) expressed specifically in high S/TFE data set.
Figure 6
Figure 6. NF-κB-signalling in stem-like human prostate TICs.
Western blots of whole cell extracts of the parent tumour, spheres and the sphere tumour analyzing (a) various signalling pathway components, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) representing loading control. (b) Nuclear localization of NF-κB in stem-like sphere-forming cells. Immunofluorescence (scale bar, 100 μm) or immunohistochemistry (scale bar, 50 μm) of acetylated (K310) NF-κB visualized in the nucleus and counterstained by DAPI (IF) or hematoxylin (IHC). (c) Comparative immuno-histochemical analysis of MCL-1 expression in primary spheres versus the parent tumour, sphere tumour and human patient primary tumour. Scale bar, 100 μm. (d) Dose-dependent effects of small molecule inhibitors on secondary sphere formation. Inhibitors were administered to primary sphere-forming total tumour cells. Red, PHA (PHA 665752); brown, parthenolide; black, 481407; green, celastrol. (e) Preliminary screening to test effect of inhibitors of different signalling pathways on secondary sphere-formation in vitro. PHA (10ìμM), parthenolide (10ìM), 481407(5ìM), celastrol (2ìM), BAY (BAY11-7082, 10ìM), staurosporine (0.05ìM), MG132 (0.5ìM), LY (LY294002, 10ìM) + rapamycin (20nM), cyclopamine (2ìM) or DAPT (10ìM) were administered, wherein the control represents dimethylsulphoxide (DMSO)-treated set. (f) Primary sphere-forming tumour cells were treated with the inhibitors of MET (PHA: 20 μM), activated NF-κB signalling, (parthenolide: 20 μM; 481407: 20 μM; celastrol: 3 μM), PI3 kinase signalling (LY 294002: 15 μM)+mTOR signalling (rapamycin: 40nM) or Notch signalling (DAPT: 20 μM) were xeno-transplanted subcutaneously. Tumour size was determined by tumour volume at the 5-week end point. (g, h) Effects of these signalling inhibitors on secondary sphere formation (g) and tumour-initiation (h) across multiple human prostate tumour xenograft models. Same number of total tumour cells from human prostate DU-145 SC-(blue)/ PC-82 OT-(red) tumours or prospectively purified TRA-1-60-positive cells from the CWR22 OT-(green)/DU-145 SC-(pink) tumours were used. Tumour size was determined as above. (i) Immunoblot analyses of cleared caspase (Cl-caspase) and PARP cleavage in the primary spheres following administration of NF-κB and AKT/mTOR signalling inhibitors. Whole cell extracts prepared 6 h-post drug administration were analysed by western blotting. Control represents DMSO-treated set, whereas the total AKT and total S6RP levels served as loading controls. Mean±s.d., n≥4.
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