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).

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:BACKGROUND:New pharmacologic targets are urgently needed to treat or prevent lung cancer, the most common cause of cancer death for men and women. This study identified one such target. This is the canonical Wnt signaling pathway, which is deregulated in cancers, including those lacking adenomatous polyposis coli or ?-catenin mutations. Two poly-ADP-ribose polymerase (PARP) enzymes regulate canonical Wnt activity: tankyrase (TNKS) 1 and TNKS2. These enzymes poly-ADP-ribosylate (PARsylate) and destabilize axin, a key component of the ?-catenin phosphorylation complex. METHODS:This study used comprehensive gene profiles to uncover deregulation of the Wnt pathway in murine transgenic and human lung cancers, relative to normal lung. Antineoplastic consequences of genetic and pharmacologic targeting of TNKS in murine and human lung cancer cell lines were explored, and validated in vivo in mice by implantation of murine transgenic lung cancer cells engineered with reduced TNKS expression relative to controls. RESULTS:Microarray analyses comparing Wnt pathway members in malignant versus normal tissues of a murine transgenic cyclin E lung cancer model revealed deregulation of Wnt pathway components, including TNKS1 and TNKS2. Real-time PCR assays independently confirmed these results in paired normal-malignant murine and human lung tissues. Individual treatments of a panel of human and murine lung cancer cell lines with the TNKS inhibitors XAV939 and IWR-1 dose-dependently repressed cell growth and increased cellular axin 1 and tankyrase levels. These inhibitors also repressed expression of a Wnt-responsive luciferase construct, implicating the Wnt pathway in conferring these antineoplastic effects. Individual or combined knockdown of TNKS1 and TNKS2 with siRNAs or shRNAs reduced lung cancer cell growth, stabilized axin, and repressed tumor formation in murine xenograft and syngeneic lung cancer models. CONCLUSIONS:Findings reported here uncovered deregulation of specific components of the Wnt pathway in both human and murine lung cancer models. Repressing TNKS activity through either genetic or pharmacological approaches antagonized canonical Wnt signaling, reduced murine and human lung cancer cell line growth, and decreased tumor formation in mouse models. Taken together, these findings implicate the use of TNKS inhibitors to target the Wnt pathway to combat lung cancer.

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A comparative reverse docking strategy to identify potential antineoplastic targets of tea functional components and binding mode.

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A comparative reverse docking strategy to identify potential antineoplastic targets of tea functional components and binding mode.

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