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The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets.

Pode-Shakked N, Shukrun R, Mark-Danieli M, Tsvetkov P, Bahar S, Pri-Chen S, Goldstein RS, Rom-Gross E, Mor Y, Fridman E, Meir K, Simon A, Magister M, Kaminski N, Goldmacher VS, Harari-Steinberg O, Dekel B - EMBO Mol Med (2012)

Bottom Line: The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear.WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family.The existence of CIC/CSCs in WT provides new therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Stem Cell Research Institute, Edmond and LiliSafra Children's Hospital, Sheba Medical Center, Ramat-Gan, Israel.

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Specific regulatory pathways in NCAM+ALDH1+ WT CICs provide insight into WT oncogenesisProteasomal levels were examined in a primary tumour (1) compared to un-sorted p-WT Xn (2) and p-WT Xn generated from NCAM+ALDH1+ cells (3), showing increased levels in the latter. Proteasomal complexes were analysed by (a) nondenaturing page analysis and total proteasomal subunit protein levels and by (b) immunoblot analysis of polyacrylamide-SDS gel.Immunoblot analysis of polyacrylamide–SDS gel for the expression of the following proteins: AKTpSer473, p-ERK, c-FOS, c-JUN, β-Catenin, NQO1, p53, c-MYC, SRC and YAP1 in parental tumour (1), NCAM+ALDH1+ derived Xn (2), foetal kidney (3) and rhabdoid tumour (4). Showing that AKTpSer473, but not p-ERK and c-FOS, are differentially expressed in NCAM+ALDH1+ derived p-WT Xn.Agilent microRNA microarray comparison of un-sorted p-WT Xn, NCAM+ALDH1+ derived p-WT Xn and human foetal kidneys. (a) Heat map of 71 miRNAs differentially expressed between p-WT Xns and human foetal kidneys. Expression pattern was similar among all p-WT Xns, with 35 down-regulated and 36 up-regulated microRNAs (miRs) compared to the hFK tissues. (b) Expression of microRNA families let-7 and 200 (miRs 200a and 200b), miR-204 and miR-337 is markedly reduced in NCAM+ALDH1+ derived p-WT Xn compared to un-sorted p-WT Xn and human foetal kidney.Suggested schematic representation of the regulatory pathways governing WT CIC phenotype and function. For array experiments four sources (n = 4) for each tissue type were used.
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fig04: Specific regulatory pathways in NCAM+ALDH1+ WT CICs provide insight into WT oncogenesisProteasomal levels were examined in a primary tumour (1) compared to un-sorted p-WT Xn (2) and p-WT Xn generated from NCAM+ALDH1+ cells (3), showing increased levels in the latter. Proteasomal complexes were analysed by (a) nondenaturing page analysis and total proteasomal subunit protein levels and by (b) immunoblot analysis of polyacrylamide-SDS gel.Immunoblot analysis of polyacrylamide–SDS gel for the expression of the following proteins: AKTpSer473, p-ERK, c-FOS, c-JUN, β-Catenin, NQO1, p53, c-MYC, SRC and YAP1 in parental tumour (1), NCAM+ALDH1+ derived Xn (2), foetal kidney (3) and rhabdoid tumour (4). Showing that AKTpSer473, but not p-ERK and c-FOS, are differentially expressed in NCAM+ALDH1+ derived p-WT Xn.Agilent microRNA microarray comparison of un-sorted p-WT Xn, NCAM+ALDH1+ derived p-WT Xn and human foetal kidneys. (a) Heat map of 71 miRNAs differentially expressed between p-WT Xns and human foetal kidneys. Expression pattern was similar among all p-WT Xns, with 35 down-regulated and 36 up-regulated microRNAs (miRs) compared to the hFK tissues. (b) Expression of microRNA families let-7 and 200 (miRs 200a and 200b), miR-204 and miR-337 is markedly reduced in NCAM+ALDH1+ derived p-WT Xn compared to un-sorted p-WT Xn and human foetal kidney.Suggested schematic representation of the regulatory pathways governing WT CIC phenotype and function. For array experiments four sources (n = 4) for each tissue type were used.

Mentions: We next determined that biological properties of the NCAM+ALDH1+ CIC/CSCs in WT. Since Xn tumours initiated from NCAM+ cells differentially expressed specific proteins such as AKTpSer473 (Fig 2E and Supporting Information Table S4), the protein profile of tumour Xn early after initiation by NCAM+ALDH1+ cells might provide insights into CIC/CSCs biology. Nondenaturing PAGE and immunoblot analysis for the expression of proteasomal subunits 19s and 20s revealed overexpression of the subunits in comparison to both tumour xenografts generated from unsorted p-WT Xn cells and parental WT (Fig 4A). Moreover, Western blot analysis demonstrated differential protein expression in the NCAM+ALDH1+ tumour, specifically AKTpSer473 but not p-ERK and c-FOS, which have been recently implicated in WT and were strongly expressed in the primary parental WT (Fig 4B). c-Jun seemed to be activated in WT in general while β-catenin was similarly observed in NCAM+ALDH1+ tumour and hFK and therefore does not necessarily specify CIC pathways. c-MYC, SRC and YAP1 were expressed in most samples (WT and control rhabdoid tumours) with c-MYC elevated in NCAM+ALDH1+ tumour. Other proteins, such as NQO1, and to a lesser extent P53, were increased in control paediatric rhabdoid tumour, further indicating the specificity of the pathways active in WT and importantly in the NCAM+ALDH1+ CIC derived tumour xenografts. Thus, highly enriched WT CIC/CSCs are likely to feature signaling molecules and oncogenes distinct from those generally observed in WT and other closely related paediatric tumours.


The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets.

Pode-Shakked N, Shukrun R, Mark-Danieli M, Tsvetkov P, Bahar S, Pri-Chen S, Goldstein RS, Rom-Gross E, Mor Y, Fridman E, Meir K, Simon A, Magister M, Kaminski N, Goldmacher VS, Harari-Steinberg O, Dekel B - EMBO Mol Med (2012)

Specific regulatory pathways in NCAM+ALDH1+ WT CICs provide insight into WT oncogenesisProteasomal levels were examined in a primary tumour (1) compared to un-sorted p-WT Xn (2) and p-WT Xn generated from NCAM+ALDH1+ cells (3), showing increased levels in the latter. Proteasomal complexes were analysed by (a) nondenaturing page analysis and total proteasomal subunit protein levels and by (b) immunoblot analysis of polyacrylamide-SDS gel.Immunoblot analysis of polyacrylamide–SDS gel for the expression of the following proteins: AKTpSer473, p-ERK, c-FOS, c-JUN, β-Catenin, NQO1, p53, c-MYC, SRC and YAP1 in parental tumour (1), NCAM+ALDH1+ derived Xn (2), foetal kidney (3) and rhabdoid tumour (4). Showing that AKTpSer473, but not p-ERK and c-FOS, are differentially expressed in NCAM+ALDH1+ derived p-WT Xn.Agilent microRNA microarray comparison of un-sorted p-WT Xn, NCAM+ALDH1+ derived p-WT Xn and human foetal kidneys. (a) Heat map of 71 miRNAs differentially expressed between p-WT Xns and human foetal kidneys. Expression pattern was similar among all p-WT Xns, with 35 down-regulated and 36 up-regulated microRNAs (miRs) compared to the hFK tissues. (b) Expression of microRNA families let-7 and 200 (miRs 200a and 200b), miR-204 and miR-337 is markedly reduced in NCAM+ALDH1+ derived p-WT Xn compared to un-sorted p-WT Xn and human foetal kidney.Suggested schematic representation of the regulatory pathways governing WT CIC phenotype and function. For array experiments four sources (n = 4) for each tissue type were used.
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fig04: Specific regulatory pathways in NCAM+ALDH1+ WT CICs provide insight into WT oncogenesisProteasomal levels were examined in a primary tumour (1) compared to un-sorted p-WT Xn (2) and p-WT Xn generated from NCAM+ALDH1+ cells (3), showing increased levels in the latter. Proteasomal complexes were analysed by (a) nondenaturing page analysis and total proteasomal subunit protein levels and by (b) immunoblot analysis of polyacrylamide-SDS gel.Immunoblot analysis of polyacrylamide–SDS gel for the expression of the following proteins: AKTpSer473, p-ERK, c-FOS, c-JUN, β-Catenin, NQO1, p53, c-MYC, SRC and YAP1 in parental tumour (1), NCAM+ALDH1+ derived Xn (2), foetal kidney (3) and rhabdoid tumour (4). Showing that AKTpSer473, but not p-ERK and c-FOS, are differentially expressed in NCAM+ALDH1+ derived p-WT Xn.Agilent microRNA microarray comparison of un-sorted p-WT Xn, NCAM+ALDH1+ derived p-WT Xn and human foetal kidneys. (a) Heat map of 71 miRNAs differentially expressed between p-WT Xns and human foetal kidneys. Expression pattern was similar among all p-WT Xns, with 35 down-regulated and 36 up-regulated microRNAs (miRs) compared to the hFK tissues. (b) Expression of microRNA families let-7 and 200 (miRs 200a and 200b), miR-204 and miR-337 is markedly reduced in NCAM+ALDH1+ derived p-WT Xn compared to un-sorted p-WT Xn and human foetal kidney.Suggested schematic representation of the regulatory pathways governing WT CIC phenotype and function. For array experiments four sources (n = 4) for each tissue type were used.
Mentions: We next determined that biological properties of the NCAM+ALDH1+ CIC/CSCs in WT. Since Xn tumours initiated from NCAM+ cells differentially expressed specific proteins such as AKTpSer473 (Fig 2E and Supporting Information Table S4), the protein profile of tumour Xn early after initiation by NCAM+ALDH1+ cells might provide insights into CIC/CSCs biology. Nondenaturing PAGE and immunoblot analysis for the expression of proteasomal subunits 19s and 20s revealed overexpression of the subunits in comparison to both tumour xenografts generated from unsorted p-WT Xn cells and parental WT (Fig 4A). Moreover, Western blot analysis demonstrated differential protein expression in the NCAM+ALDH1+ tumour, specifically AKTpSer473 but not p-ERK and c-FOS, which have been recently implicated in WT and were strongly expressed in the primary parental WT (Fig 4B). c-Jun seemed to be activated in WT in general while β-catenin was similarly observed in NCAM+ALDH1+ tumour and hFK and therefore does not necessarily specify CIC pathways. c-MYC, SRC and YAP1 were expressed in most samples (WT and control rhabdoid tumours) with c-MYC elevated in NCAM+ALDH1+ tumour. Other proteins, such as NQO1, and to a lesser extent P53, were increased in control paediatric rhabdoid tumour, further indicating the specificity of the pathways active in WT and importantly in the NCAM+ALDH1+ CIC derived tumour xenografts. Thus, highly enriched WT CIC/CSCs are likely to feature signaling molecules and oncogenes distinct from those generally observed in WT and other closely related paediatric tumours.

Bottom Line: The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear.WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family.The existence of CIC/CSCs in WT provides new therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Stem Cell Research Institute, Edmond and LiliSafra Children's Hospital, Sheba Medical Center, Ramat-Gan, Israel.

Show MeSH
Related in: MedlinePlus