Project description:BACKGROUND: Several in vitro assays have been used to identify “cancer stem cells” (CSC), including expression of cell surface markers and Hoechst dye efflux properties. However, each of these methods has potential pitfalls that complicate interpretation of the results. Focusing on colon cancers (CC), the CD133 antigen has been proposed as a marker of colon CSC. However, conflicting results have been reported in the literature indicating the need of a systematic analysis of CSC within CC and a complete validation of markers for the isolation of these cells. AIMS: Aim of this study was to confirm that CD133 expression is a valid method for isolating CSC in CC and verify if other antigens can increase the specificity of this marker for isolating CSC in CC. METHODS: CD133+ and CD133- cells were isolated from different human CC lines (CaCo-2, HT29, LOVO, HCT-116) by FACS sorter and the tumor-initiating potential of CD133+ cells was assessed in vitro, by soft-agar colony formation assay, and in vivo, upon transplantation into nude mice. Furthermore, the gene expression profile of CD133+ versus CD133- CaCo-2 cells was compared by the means of microarray analysis. Then, in the effort to identify a common “tumor stem cell” signature for CC, the most relevant transcripts resulting from gene expression profiling on CD133+ cells was assessed by real-time PCR on SP-fraction isolated by FACS sorter from the same CC cell lines. Finally, we deplete CD133 expression in the CaCo-2 cell line by the means of siRNA and verified by Western Blot analysis whether there was a functional correlation between CD133 and the target genes. Moreover, CaCo-2 and HCT116 cells were exposed to sodium butyrate (NaBu) for 72h. Colon cells differentiation was assessed by Alkaline phosphatase activity and expression of CD133 and target genes was tested by western blot. RESULTS: We confirmed that only CD133+ cells have a tumor-initiating potential in vitro and in vivo. Furthermore, microarray analysis of CD133+ versus CD133- CaCo-2 cells revealed a significant overexpression of various transcripts involved in cell proliferation, invasion and stemness in CD133+ cell fraction. Comparison of the transcripts by real-time PCR revealed that the genes of Endothelin-1 (END-1) and NR4A2 are highly expressed in both CD133 + cells and in SP fractions. Finally, when we deplete CD133 expression in Caco-2cells by siRNA, we observed a significant attenuation of END-1 and NR4A2 expression, thus demonstrating that CD133 is involved in the transcriptional regulation of these genes. Interestingly, we also showed that the expression of all three genes was inversely correlated with cell differentiation status as demonstrated by the fact that their expression decreases in a time- and dose-dependent manner after differentiation induced by NaBu. CONCLUSION: Overall, this study confirms the role of CD133antigen as CSC marker and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression, hypothesizing that CD133 is involved in the transcriptional regulation of these gene. Microarray analysis was performed on CD133+ and CD133- sorted CACO-2 cells. For both fractions, cells were sorted three independent times. Sample preparation was performed according to Affymetrix recommendations. A total of 6 arrays were hybridized, including 3 CD133+ replicates and 3 CD133- replicates.

Project description:BACKGROUND: Several in vitro assays have been used to identify “cancer stem cells” (CSC), including expression of cell surface markers and Hoechst dye efflux properties. However, each of these methods has potential pitfalls that complicate interpretation of the results. Focusing on colon cancers (CC), the CD133 antigen has been proposed as a marker of colon CSC. However, conflicting results have been reported in the literature indicating the need of a systematic analysis of CSC within CC and a complete validation of markers for the isolation of these cells. AIMS: Aim of this study was to confirm that CD133 expression is a valid method for isolating CSC in CC and verify if other antigens can increase the specificity of this marker for isolating CSC in CC. METHODS: CD133+ and CD133- cells were isolated from different human CC lines (CaCo-2, HT29, LOVO, HCT-116) by FACS sorter and the tumor-initiating potential of CD133+ cells was assessed in vitro, by soft-agar colony formation assay, and in vivo, upon transplantation into nude mice. Furthermore, the gene expression profile of CD133+ versus CD133- CaCo-2 cells was compared by the means of microarray analysis. Then, in the effort to identify a common “tumor stem cell” signature for CC, the most relevant transcripts resulting from gene expression profiling on CD133+ cells was assessed by real-time PCR on SP-fraction isolated by FACS sorter from the same CC cell lines. Finally, we deplete CD133 expression in the CaCo-2 cell line by the means of siRNA and verified by Western Blot analysis whether there was a functional correlation between CD133 and the target genes. Moreover, CaCo-2 and HCT116 cells were exposed to sodium butyrate (NaBu) for 72h. Colon cells differentiation was assessed by Alkaline phosphatase activity and expression of CD133 and target genes was tested by western blot. RESULTS: We confirmed that only CD133+ cells have a tumor-initiating potential in vitro and in vivo. Furthermore, microarray analysis of CD133+ versus CD133- CaCo-2 cells revealed a significant overexpression of various transcripts involved in cell proliferation, invasion and stemness in CD133+ cell fraction. Comparison of the transcripts by real-time PCR revealed that the genes of Endothelin-1 (END-1) and NR4A2 are highly expressed in both CD133 + cells and in SP fractions. Finally, when we deplete CD133 expression in Caco-2cells by siRNA, we observed a significant attenuation of END-1 and NR4A2 expression, thus demonstrating that CD133 is involved in the transcriptional regulation of these genes. Interestingly, we also showed that the expression of all three genes was inversely correlated with cell differentiation status as demonstrated by the fact that their expression decreases in a time- and dose-dependent manner after differentiation induced by NaBu. CONCLUSION: Overall, this study confirms the role of CD133antigen as CSC marker and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression, hypothesizing that CD133 is involved in the transcriptional regulation of these gene.

Project description:Metastatic melanoma is difficult to treat and is resistant to most current therapies. We hypothesized that stem cell related pathways may play a role in melanoma growth since stem cells and cancer cells share essential properties. Lately, the ABC transporter, ABCB5, as well as Cripto-1, CD20, CD133 and ABCG2 were used to isolate tumorigenic cells. Melanoma cells expressing MDR1 (ABCB1) were found in our lab to be enriched with ABCB5, Nanog, and Oct3/4 expression and were characterized by increased self-renewal capacity and anchorage independence. Melanoma significant heterogeneity was further demonstrated by Quintana et al. who found high prevalence of melanoma initiating cells when implanted into severely immunocompromised mice. Therefore, we took a more general approach in order to unravel pathways that are important for self-renewal and tumorigenicity of melanoma. We enriched for melanoma precursor cells (MPC) using culture conditions shown to enrich for normal melanocyte precursors. MPCs expressed higher levels of nanog and Oct3/4, as well as ABCB1 (MDR1) and ABCB5. Microarray analysis revealed deregulation of genes related to development, invasion, and cell cycle, in addition to upregulation of genes involved in Wnt signaling such as DVL3, DKK1, ID3, JAG1 and Wnt receptor, FZD7. We focused on Wnt signaling and found enrichment of FZD7+ cells in MPCs. Furthermore, FZD7+ cells co-expressed MDR1 and were enriched in highly tumorigenic xenograft parts when tested in mice. Inhibition of the Wnt pathway using secreted frizzled related protein-1 (sFRP1), resulted in attenuation of melanoma proliferation in a dose dependent manner demonstrating the important role of the Wnt pathway in melanoma growth and suggesting this pathway as a target for rational therapy. The canonical Wnt/beta-catenin pathway was not activated in MPC, as revealed using the Tcf Reporter assay. In contrast, we found increased of phosphorylated CAMKII, suggesting activation of the non-canonical Wnt pathway. Our data indicate that precursors within the melanoma tumor may acquire increased survival abilities that should be therapeutically targeted. In particular, we found that the non canonical wnt pathway and the wnt receptor FZD7 play a role in melanoma proliferation and tumorigenicity. We used specialized culture conditions that were found to enrich for normal melanoblasts, the precursors of melanocytes [7]. We found previously that these conditions result in a typical dedifferentiated unpigmented phenotype of melanoma cells in addition to enrichment of self-renewing cells [2]. Further characterization of these precursor cultures prepared from 6 human metastatic melanoma lesions revealed increased expression of stemness genes such as nanog and Oct3/4, as well as two recently reported melanoma precursor markers, MDR1 and ABCB5. Consistent with the normal melanoblast cultures we found increased expression of the melanoblast marker POU3F2 (BRN2, DCT). In contrast, the expression of MITF, a melanocyte differentiation marker was reduced by 85% in MPCs when compared with the primary culture (Figure 1a, relative expression of MPC vs. cells obtained from the same patient and cultured in regular conditions). Cell cycle analysis excluded death of differentiated cells as the cause of the precursor enrichment, as no apoptosis was evident (Figure 1b, c). Interestingly, there was an increase in S phase cells, indicating that these cells are undergoing divisions. Similarly, Wang et al. reported that CD133+ glioma cells have higher percentage of S phase cells as compared to CD133- cells [8]. We concluded that these culture conditions cause dedifferentiation of melanocytes in addition to enrichment by increased divisions of melanoma precursor cells. Therefore, we defined these cells as melanoma precursor cells (MPC). To reveal pathways of self-renewal in melanoma we have used two single cell clones that were prepared from primary human melanoma cultures obtained from a primary lesion. These cell clones differed in the self-renewal capacity (in the limiting dilution assay) and in their morphology: high self-renewal/polygonal vs low self-renewal/spindle shaped. We compared the two cell types described above, cultured in either regular RPMI-based media or culture conditions developed to enrich for normal melanocytic precursors (Cook AL, Donatien PD, Smith AG, Murphy M, Jones MK, et al. (2003) Human melanoblasts in culture: expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3. J Invest Dermatol 121: 1150-1159.), designated MPC (melanoma precursor cells) conditions, for two weeks.

Project description:Metastatic melanoma is difficult to treat and is resistant to most current therapies. We hypothesized that stem cell related pathways may play a role in melanoma growth since stem cells and cancer cells share essential properties. Lately, the ABC transporter, ABCB5, as well as Cripto-1, CD20, CD133 and ABCG2 were used to isolate tumorigenic cells. Melanoma cells expressing MDR1 (ABCB1) were found in our lab to be enriched with ABCB5, Nanog, and Oct3/4 expression and were characterized by increased self-renewal capacity and anchorage independence. Melanoma significant heterogeneity was further demonstrated by Quintana et al. who found high prevalence of melanoma initiating cells when implanted into severely immunocompromised mice. Therefore, we took a more general approach in order to unravel pathways that are important for self-renewal and tumorigenicity of melanoma. We enriched for melanoma precursor cells (MPC) using culture conditions shown to enrich for normal melanocyte precursors. MPCs expressed higher levels of nanog and Oct3/4, as well as ABCB1 (MDR1) and ABCB5. Microarray analysis revealed deregulation of genes related to development, invasion, and cell cycle, in addition to upregulation of genes involved in Wnt signaling such as DVL3, DKK1, ID3, JAG1 and Wnt receptor, FZD7. We focused on Wnt signaling and found enrichment of FZD7+ cells in MPCs. Furthermore, FZD7+ cells co-expressed MDR1 and were enriched in highly tumorigenic xenograft parts when tested in mice. Inhibition of the Wnt pathway using secreted frizzled related protein-1 (sFRP1), resulted in attenuation of melanoma proliferation in a dose dependent manner demonstrating the important role of the Wnt pathway in melanoma growth and suggesting this pathway as a target for rational therapy. The canonical Wnt/beta-catenin pathway was not activated in MPC, as revealed using the Tcf Reporter assay. In contrast, we found increased of phosphorylated CAMKII, suggesting activation of the non-canonical Wnt pathway. Our data indicate that precursors within the melanoma tumor may acquire increased survival abilities that should be therapeutically targeted. In particular, we found that the non canonical wnt pathway and the wnt receptor FZD7 play a role in melanoma proliferation and tumorigenicity. We used specialized culture conditions that were found to enrich for normal melanoblasts, the precursors of melanocytes [7]. We found previously that these conditions result in a typical dedifferentiated unpigmented phenotype of melanoma cells in addition to enrichment of self-renewing cells [2]. Further characterization of these precursor cultures prepared from 6 human metastatic melanoma lesions revealed increased expression of stemness genes such as nanog and Oct3/4, as well as two recently reported melanoma precursor markers, MDR1 and ABCB5. Consistent with the normal melanoblast cultures we found increased expression of the melanoblast marker POU3F2 (BRN2, DCT). In contrast, the expression of MITF, a melanocyte differentiation marker was reduced by 85% in MPCs when compared with the primary culture (Figure 1a, relative expression of MPC vs. cells obtained from the same patient and cultured in regular conditions). Cell cycle analysis excluded death of differentiated cells as the cause of the precursor enrichment, as no apoptosis was evident (Figure 1b, c). Interestingly, there was an increase in S phase cells, indicating that these cells are undergoing divisions. Similarly, Wang et al. reported that CD133+ glioma cells have higher percentage of S phase cells as compared to CD133- cells [8]. We concluded that these culture conditions cause dedifferentiation of melanocytes in addition to enrichment by increased divisions of melanoma precursor cells. Therefore, we defined these cells as melanoma precursor cells (MPC).

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Knockdown of CD133 in melanoma cells

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