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

Rajasekhar VK, Studer L, Gerald W, Socci ND, Scher HI - Nat Commun (2011)

Bottom Line: 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.These TICs exhibit increased nuclear factor-κB activity.

View Article: PubMed Central - PubMed

Affiliation: 1] Stem Cell Center and Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. [2] Sidney Kimmel Center for Prostate and Urologic Cancers, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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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f6: 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.

Mentions: We investigated the differences between the TRA-1-60-positive spheres with tumour-initiating potential and tumours with regards to various signalling pathways mediated by canonical receptor tyrosine kinase, insulin, AKT, ERK and Wnt (for example, β-catenin expression). Except for a diminished level of insulin growth factor receptor 1β phosphorylation in the spheres (Fig. 6a; Supplementary Fig. S1a,b,d), there were no considerable differences in the activation of conventionally studied signalling pathway components. Therefore, we reasoned from genome-wide expression data that constitutively active PKC/NF-κB pathway may be functionally associated with the stem-like human prostate TICs. We verified that a classical PKC (PKC α) was specifically activated in the stem-like TICs by immunoblotting for PKC α protein phosphorylation (Fig. 6a). However, levels of other PKCs (pan-phosphorylated PKCβII and atypical PKCs, PKC ζ/λ) remained unaffected, whereas the PKD or PKCμ remained moderately lower in the sphere cells as compared with the tumour cells. Next, we confirmed the microarray data on downregulation of transcription of NFKBIA (Fig. 5c) with a specific decrease in its protein level, by including the other positive controls such as IGFBP7 and αB-crystallin (Figs 1h and 6a). There was also an increased accumulation of NF-κB p65 and its K310 acetylated form specifically nuclear localized in the spheres (Fig. 6b), implying a potential functional importance of sustained NF-κB signalling, as deacetylation of NF-κB p65 was previously shown to inhibit its transcriptional activity and induce cellular apoptosis32. Furthermore, the sphere cells showed increased levels of a proinflammatory cytokine interleukin 6 (IL-6), the anti-apoptotic Bcl-2 family member human myeloid cell leukemia-1 (MCL-1) and additional regulators of epithelial cell proliferation and apoptosis, such as 14-3-3σ (Fig. 6a,c).


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

Rajasekhar VK, Studer L, Gerald W, Socci ND, Scher HI - Nat Commun (2011)

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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Related In: Results  -  Collection

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f6: 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.
Mentions: We investigated the differences between the TRA-1-60-positive spheres with tumour-initiating potential and tumours with regards to various signalling pathways mediated by canonical receptor tyrosine kinase, insulin, AKT, ERK and Wnt (for example, β-catenin expression). Except for a diminished level of insulin growth factor receptor 1β phosphorylation in the spheres (Fig. 6a; Supplementary Fig. S1a,b,d), there were no considerable differences in the activation of conventionally studied signalling pathway components. Therefore, we reasoned from genome-wide expression data that constitutively active PKC/NF-κB pathway may be functionally associated with the stem-like human prostate TICs. We verified that a classical PKC (PKC α) was specifically activated in the stem-like TICs by immunoblotting for PKC α protein phosphorylation (Fig. 6a). However, levels of other PKCs (pan-phosphorylated PKCβII and atypical PKCs, PKC ζ/λ) remained unaffected, whereas the PKD or PKCμ remained moderately lower in the sphere cells as compared with the tumour cells. Next, we confirmed the microarray data on downregulation of transcription of NFKBIA (Fig. 5c) with a specific decrease in its protein level, by including the other positive controls such as IGFBP7 and αB-crystallin (Figs 1h and 6a). There was also an increased accumulation of NF-κB p65 and its K310 acetylated form specifically nuclear localized in the spheres (Fig. 6b), implying a potential functional importance of sustained NF-κB signalling, as deacetylation of NF-κB p65 was previously shown to inhibit its transcriptional activity and induce cellular apoptosis32. Furthermore, the sphere cells showed increased levels of a proinflammatory cytokine interleukin 6 (IL-6), the anti-apoptotic Bcl-2 family member human myeloid cell leukemia-1 (MCL-1) and additional regulators of epithelial cell proliferation and apoptosis, such as 14-3-3σ (Fig. 6a,c).

Bottom Line: 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.These TICs exhibit increased nuclear factor-κB activity.

View Article: PubMed Central - PubMed

Affiliation: 1] Stem Cell Center and Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. [2] Sidney Kimmel Center for Prostate and Urologic Cancers, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

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.

No MeSH data available.


Related in: MedlinePlus