Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy.
All models discussed here have their defined strengths, but also limitations with respect to their predictive features. Understanding the preclinical models in a more profound way should lead to optimized clinical trials, thereby expanding the therapeutic arsenal and improving patient outcome further.
The E3 ubiquitin ligase and tumor suppressor APC/CCdh1 is crucial for cell cycle progression, development and differentiation in many cell types. However, little is known about the role of Cdh1 in hematopoiesis. Here we analyzed Cdh1 expression and function in malignant hematopoiesis. We found a significant decrease of Cdh1 in primary acute myeloid leukemia (AML) blasts compared to normal CD34+ cells. Thus, according to its important role in connecting cell cycle exit and differentiation, decreased expression of Cdh1 may be a mechanism contributing to the differentiation block in leukemogenesis. Indeed, knockdown (kd) of Cdh1 in HL-60 cell line (AML with maturation, FAB M2) led to less differentiated cells and a delay in PMA-induced differentiation. Acute promyelocytic leukemia (APL, FAB M3) is an AML subtype which is highly vulnerable to differentiation therapy with all-trans retinoic acid (ATRA). Accordingly, we found that APL is resistant to a Cdh1-kd mediated differentiation block. However, further depletion of Cdh1 in APL significantly reduced viability of leukemia cells upon ATRA-induced differentiation. Thus, low Cdh1 expression may be important in AML biology by contributing to the differentiation block and response to therapy depending on differences in the microenvironment and the additional genetic background.
The E2F1 transcription factor enhances apoptosis by DNA damage in tumors lacking p53. To elucidate the mechanism of a potential cooperation between E2F1 and chemotherapy, whole-genome microarrays of chemoresistant tumor cell lines were performed focusing on the identification of cooperation response genes (CRG). This gene class is defined by a synergistic expression response upon endogenous E2F1 activation and drug treatment. Cluster analysis revealed an expression pattern of CRGs similar to E2F1 mono-therapy, suggesting that chemotherapeutics enhance E2F1-dependent gene expression at the transcriptional level. Using this approach as a tool to explore E2F1-driven gene expression in response to anticancer drugs, we identified novel apoptosis genes such as the tumor suppressor TIEG1/KLF10 as direct E2F1 targets. We show that TIEG1/KLF10 is transcriptionally activated by E2F1 and crucial for E2F1-mediated chemosensitization of cancer cells. Our results provide a broader picture of E2F1-regulated genes in conjunction with cytotoxic treatment that allows the design of more rational therapeutics.
Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo. Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice. Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation. Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.
1236 Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation to all types of blood lineages. These unique processes are regulated by intrinsic and extrinsic signals (e.g. cytokines, cell-cell contacts) and strongly connects stem cell fate with the cell cycle. The ubiquitin-proteasome system regulates spatial and temporal abundance of proteins in the cell. During cell cycle, the anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks proteins for proteasomal degradation and thus controls their activity. Known targets of Cdh1, namely Skp2 and Id2, are involved in regulation of self-renewal and granulopoiesis (Wang et al., Blood 2011; Buitenhuis et al., Blood 2005). This raises the hypothesis that Cdh1 may be a critical upstream regulator of HSC differentiation. The analysis of human bone marrow cell subsets (CD34+, lymphoid and myeloid cells) revealed highest protein level of Cdh1 in CD34+ cells, lower expression in more mature lymphoid subsets (CD3+, CD19+) and only marginal expression in mature myeloid cells (CD41a+, CD11b+). These data suggest that Cdh1 is important to induce differentiation, but dispensable for maintaining the differentiated state. In vitro cultivation of G-CSF mobilized peripheral blood CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed downregulation of Cdh1 during culture compared to d0. Western blots did not only reveal decreasing levels of Cdh1, but also its inactivation by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the cyclin-dependent-kinase inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. To analyze cell division/proliferation and self-renewal versus differentiation more closely, we used the fluorescent dye CFSE as an indicator of cell division in combination with CD34 to indicate the differentiation status. When cultured under self-renewal conditions using SCF, TPO and Flt3-l, CD34+cells showed enhanced proliferation with increased cells in higher generations, whereas using SCF and G-CSF to induce granulopoiesis, cells within lower generations were more prominent. These experiments also revealed a rapid decrease of CD34 expression in granulopoiesis after 3 cell divisions in contrast to a moderate decline under self-renewal conditions. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual cell cycle exit and differentiation under conditions that induce granulopoiesis. To further elucidate the role of Cdh1 for stem/progenitor cell fate, we used a lentiviral knockdown of Cdh1 in CD34+ cells. After 4 days of transduction and cell sorting, the cells were cultivated for 1 week in medium containing SCF, TPO and Flt3-l. Cdh1 depleted cells showed enhanced proliferation compared to the empty vector control and a higher expression of CD34. In colony forming unit (CFU) assays, we observed that CD34+ cells with Cdh1-knockdown were less efficient to differentiate to CFU-G, CFU-M and BFU-E. A higher potential to self-renew was validated by replating of these colonies, where the number with Cdh1-knockdown increased during serial replating. To validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain−/− (NSG) xenotransplant mouse model. The evaluation of engraftment capacity and differentiation potential of human Cdh1 depleted CD34+ cells in this model is ongoing. Our data establish the central cell cycle regulator APC/CCdh1 as a novel regulator of self-renewal and differentiation in CD34+ HSCs. Disclosures: No relevant conflicts of interest to declare.
Introduction Cdh1 is an important activator of the anaphase-promoting complex/cyclosome (APC/C) and may play a major role in both the stabilization of G1-phase and the induction of cell cycle arrest and differentiation. Our work focuses on the function of APC/CCdh1in hematopoietic stem cells (HSCs) with regard to their potential for differentiation, self-renewal and malignant transformation. Methods Physiological expression levels of Cdh1 were studied among different human hematopoietic lineages (defined by staining for the cell surface markers CD11b, CD41a, CD34, CD3 and CD19) obtained from bone marrow (BM) of healthy donors and mobilized peripheral blood (PB). Next we established a strong lentiviral Cdh1 knock down (kd) in human CD34+ cells and performed colony forming cell (CFC) assays and replating assays. We also analyzed Cdh1-protein levels in 30 samples of BM or PB of patients first diagnosed with acute myeloid leukemia (AML). Finally experiments to further look into possible mechanisms of Cdh1 regulation during leukemogenesis were carried out. On the transcriptional level we reanalyzed published microarray data from CD34+-AML blasts and normal CD34+ cells (Leukemia 2011;25:1825-1833). On the post-transcriptional level we tested the hypothesis of Cdh1 degradation mediated by the ubiquitin-ligase SCF by expressing a dominant negative mutant of the core SCF subunit Cullin-1 (delta-Cul1) in the AML cell lines Kasumi-1 and HL-60. Results Western blot analysis of physiological Cdh1-distribution among the variable human hematopoietic lineages showed significant differences in Cdh1 protein levels. We saw diminishing levels of Cdh1 from HSCs to mature lymphoid and myeloid cells, suggesting that Cdh1 may be important to induce differentiation but dispensable for maintaining the differentiated state. In the Cdh1-kd-CFC assays a significant decrease of total colony numbers, CFU-Gs, CFU-GMs and BFU-Es >50% was observed. At morphological examination and FACS analysis these colonies proved to be more immature than the control colonies. Thus, depletion of Cdh1 in HSC hinders normal differentiation into the myeloid and erythroid lineage both by decreasing the number of mature lineage progenitors and by delaying individual cell maturation. Upon replating, we noticed a significant increase in the number of secondary colonies, with a doubling of total colony numbers, when using Cdh1 deficient HSC. This result indicates an advantage for self-renewal over differentiation in these cells, which seems to correlate with the intensity of the Cdh1-kd. Examination of Cdh1 protein levels in AML blasts revealed that basically all AML samples showed a strong down-regulation of Cdh1 protein levels compared to normal CD34+ cells, which may be a contributing factor to the differentiation block in leukemogenesis. Indeed, if we performed knockdown of Cdh1 in the HL-60 leukemia cell line they were in a less differentiated state as judged by CD11b expression. The evaluation of microarray data, in order to further address the mechanism of Cdh1 down-regulation in AML blasts, showed that Cdh1 transcription levels were not significantly different in CD34+ AML cells compared to normal CD34+ cells. This would be consistent with a posttranscriptional cause of decreased Cdh1-protein levels in AML blasts. Our ongoing work indicates SCF-dependent degradation of Cdh1, since inhibition of the SCF function (by expression of a dominant-negative form of the SCF subunit Cullin-1 (delta-Cul1)) in AML cell lines leads to a strong upregulation of Cdh1. Conclusions Our data establish Cdh1 as an important cell cycle regulator in the regulation of differentiation and self-renewal in HSCs. Its posttranscriptional downregulation by the SCF ubiquitin ligase may contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.
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