Dedifferentiated Liposarcoma Cell Lines Research Articles

Preclinical Evaluation of the Pan-FGFR Inhibitor LY2874455 in FRS2-Amplified Liposarcoma.

Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLPS) with amplified FRS2, but we previously only demonstrated transient cytostatic effects when treating FRS2-amplified DDLPS cells with NVP-BGJ398. Methods: Effects of the more potent FGFR inhibitor LY2874455 were investigated in three DDLPS cell lines by measuring effects on cell growth and apoptosis in vitro and also testing efficacy in vivo. Genome, transcriptome and protein analyses were performed to characterize the signaling components in the FGFR pathway. Results: LY2874455 induced a stronger, longer-lasting growth inhibitory effect and moderate level of apoptosis for two cell lines. The third cell line, did not respond to FGFR inhibition, suggesting that FRS2 amplification alone is not sufficient to predict response. Importantly, efficacy of LY2874455 was confirmed in vivo, using an independent FRS2-amplified DDLPS xenograft model. Expression of FRS2 was similar in the responding and non-responding cell lines and we could not find any major difference in downstream FGFR signaling. The only FGF expressed by unstimulated non-responding cells was the intracellular ligand FGF11, whereas the responding cell lines expressed extracellular ligand FGF2. Conclusion: Our study supports LY2874455 as a better therapy than NVP-BGJ398 for FRS2-amplified liposarcoma, and a clinical trial is warranted.

Retracted Article: miR-199a-3p knockdown inhibits dedifferentiated liposarcoma (DDLPS) cell viability and enhances apoptosis through targeting casein kinase-1 alpha (CK1α).

Dedifferentiated liposarcoma (DDLPS) is an aggressive tumor with high mortality. More insight into the biology of DDLPS tumorigenesis is needed to devise novel therapeutic approaches. Previous data showed that miRNA-199a-3p (miR-199a-3p) was strongly upregulated in DDLPS tissues. However, the biological role of miR-199a-3p in DDLPS remains unknown. In this study, we detected miR-199a-3p expression using RT-qPCR and observed that miR-199a-3p was more highly expressed in DDLPS tissues and cell lines (SW872 and LPS141). Functionally, MTT assay, flow cytometry and western blot results demonstrated that knockdown of miR-199a-3p inhibited DDLPS cell viability, enhanced apoptosis rate, and decreased expression of apoptosis-related genes Bax and cleaved caspase 3, as well as increased Bcl-2 expression in vitro. Moreover, xenograft tumors were generated and miR-199a-3p knockdown could suppress DDLPS xenograft tumor growth accompanying decreased proliferating cell nuclear antigen (PCNA) level and increased cleaved caspase 3 level in vivo. Mechanically, luciferase reporter assay and RNA immunoprecipitation (RIP) identified that CK1α was targeted and downregulated by miR-199a-3p. Expression of CK1α was lower in DDLPS tissues. Besides, there was a negative linear correlation between expressions of miR-199a-3p and CK1α in DDLPS tissues. Rescue experiments indicated that CK1α silencing could abolish the effect of miR-199a-3p knockdown on cell viability and apoptosis in DDLPS cells in vitro. In conclusion, knockdown of miR-199a-3p inhibits DDLPS cell viability and enhances apoptosis through targeting CK1α in vitro and in vivo. Our results suggest miR-199a-3p/CK1α axis may be a novel pathogen of DDLPS.

Abstract 3529: Modulation of MDM2 alters the metabolomic programming of dedifferentiated liposarcoma and its sensitivity to cholesterol inhibition

Abstract BACKGROUND: Dedifferentiated liposarcoma (DDLPS) is a highly morbid, mesenchymal tumor characterized by amplification of the 12q chromosomal loci. Although DDLPS are lipid tumors, they produce little lipid. Life expectancy is typically less than 15 months in the advanced setting and standard treatments remain highly toxic. Superior treatment options are clearly needed. Amplification of the MDM2 oncogene that resides in these loci is observed in 100% of all DDLPS; however, to variable levels. Through a negative feedback loop, MDM2 inhibits the tumor suppressive functions of p53 that halts cell growth after cellular stress. Previously, we have reported that MDM2 levels correspond with cellular growth and drug metabolism, but little is known about the effects of MDM2 alterations on global metabolomic profiles. These profiles will help us pinpoint dysregulated pathways that explain, at least partially, the functional effects associated with MDM2 amplification. METHODS: Six DDLPS cell lines were brought directly into culture from patients. MDM2 levels were determined via Western blot and RNA-sequencing. Metabolomics data was generated using the Metabolon platform. Cell viability assays were performed in ZOOM IncuCyte or measured by XTT. Atorvastatin was used to inhibit cholesterol synthesis. MDM2 levels were altered using SAR405838 to raise MDM2 levels and MI-192 to lower MDM2 levels. Synergy was calculated via Chou-Talalay method to determine the combination index (CI) using Compusyn software. RESULTS: MDM2 levels are inversely correlated with metabolites in the lipid and cholesterol pathway (Fisher's, p < 0.001). The lipid metabolism pathway was also the top deregulated pathway (pathway enrichment p-value = 0.03) in transcriptionally profiled DDLPS cell lines treated with MDM2 elevating agent SAR405838. MDM2 low DDLPS cell lines were exquisitely sensitive to HMGA-CoA reductase inhibitors in the low micromolar range. Lipid metabolite profiling of MDM2 low versus high cell lines treated with atorvastatin demonstrated that twice as many lipid metabolites were altered in MDM2 low versus high cells (Chi-square, p < 0.001). MDM2 levels by RNA-seq demonstrated significant correlation between MDM2 gene expression and atorvastatin IC50 doses (r=0.963). MDM2 modulation by use of SAR405838 and MI-192 respectively in combination with atorvastatin displayed antagonism (average CI = 1.5) and synergy (average CI = 0.63), respectively. CONCLUSION: Modulation of MDM2 alters cholesterol metabolism in DDLPS and may serve as druggable target. Citation Format: Bryce Demoret, Andrew Patt, John Hays, Ewy Mathé, James L. Chen. Modulation of MDM2 alters the metabolomic programming of dedifferentiated liposarcoma and its sensitivity to cholesterol inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3529.

MiR-133a function in the pathogenesis of dedifferentiated liposarcoma

BackgroundSarcomas are malignant heterogeneous tumors of mesenchymal derivation. Dedifferentiated liposarcoma (DDLPS) is aggressive with recurrence in 80% and metastasis in 20% of patients. We previously found that miR-133a was significantly underexpressed in liposarcoma tissues. As this miRNA has recently been shown to be a tumor suppressor in many cancers, the objective of this study was to characterize the biological and molecular consequences of miR-133a underexpression in DDLPS.MethodsReal-time PCR was used to evaluate expression levels of miR-133a in human DDLPS tissue, normal fat tissue, and human DDLPS cell lines. DDLPS cells were stably transduced with miR-133a vector to assess the effects in vitro on proliferation, cell cycle, cell death, migration, and metabolism. A Seahorse Bioanalyzer system was also used to assess metabolism in vivo by measuring glycolysis and oxidative phosphorylation (OXPHOS) in subcutaneous xenograft tumors from immunocompromised mice.ResultsmiR-133a expression was significantly decreased in human DDLPS tissue and cell lines. Enforced expression of miR-133a decreased cell proliferation, impacted cell cycle progression kinetics, decreased glycolysis, and increased OXPHOS. There was no significant effect on cell death or migration. Using an in vivo xenograft mouse study, we showed that tumors with increased miR-133a expression had no difference in tumor growth compared to control, but did exhibit an increase in OXPHOS metabolic respiration.ConclusionsBased on our collective findings, we propose that in DDPLS, loss of miR-133a induces a metabolic shift due to a reduction in oxidative metabolism favoring a Warburg effect in DDLPS tumors, but this regulation on metabolism was not sufficient to affect DDPLS.

Abstract 3426: miR-133a function in the pathogenesis of dedifferentiated liposarcoma

Abstract Background: Sarcomas are malignant heterogeneous tumors of mesenchymal derivation. Dedifferentiated liposarcoma (DDLPS) exhibits aggressive biological behavior with an 80% local recurrence rate and the development of metastasis in approximately 20% of patients. To identify and characterize the molecular alterations that contribute to the development and progression of DDLPS, our laboratory characterized a unique miRNA expression signature associated with human DDLPS tissue compared to paired subjacent normal tissue. We found that several muscle-specific miRNAs (myomiRs), miR-1, -133a, and -206, were significantly underexpressed in liposarcoma tissues. Interestingly, these miRNAs have recently been shown to have tumor suppressor functions in many human cancers. The objective of this study is to characterize the biological and molecular consequences of miR-133a in DDLPS. Methods: Taqman real-time PCR was used to evaluate expression levels of myomiRs in human DDLPS tissue, normal fat tissue, and available human DDLPS cell lines. To evaluate the effects of miR-133a expression on DDLPS cell line behavior in vitro, cells were stably transduced with miR-133a vector or empty lentiviral vector and the effects on cell cycle, proliferation, apoptosis, and migration were assessed. Agilent Seahorse Bioanalyzer system was used to assess metabolism. We performed an in silico search for predicted targets of miR-133a using target prediction databases and compared these target genes to known genes that are highly expressed in human DDLPS tissue. Results: The expression levels of myomiRs were significantly decreased in human DDLPS tissue compared to normal human fat as well as in human DDLPS cell lines compared to a human preadipocyte cell lines. Overexpression of miR-133a decreased cell proliferation, decreased glycolysis, and increased spare respiratory capacity. There was no significant effect on cell cycle, apoptosis, or migration. Using in silico target gene analysis, we identified 18 potential targets of miR-133a. These preliminary results suggest that miR-133a regulates metabolism and proliferation, and its dysregulation might contribute to the oncogenic phenotype of DDLPS. Significance: We have demonstrated that enforced expression of miR-133a decreased proliferation and metabolism in human DDLPS cells. We have identified putative gene targets of miR-133a and are currently dissecting the molecular mechanism by which miR-133a may mediate proliferation and metabolism in DDLPS cell lines. Taken together, these data suggest that miR-133a might play a tumor suppressor role in DDLPS, thereby generating new potential targets for therapeutic intervention to alter the course of this disease. Citation Format: Peter Y. Yu, Hans Iwenofu, Raphael E. Pollock, Denis C. Guttridge. miR-133a function in the pathogenesis of dedifferentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2017-3426

Activation of the Akt-mTOR and MAPK pathways in dedifferentiated liposarcomas.

The Akt/mTOR and MAPK pathways play important roles in modulating cellular function in response to extracellular signals, and they are known to be activated in certain kinds of sarcomas. Few investigations have examined these pathways in dedifferentiated liposarcoma (DDLS), in relation to clinicopathological features. Clinicopathological and immunohistochemical analyses were conducted using 99 DDLS specimens. An in vitro study was also conducted to examine the antitumor effects of an mTOR inhibitor and a MEK inhibitor on two DDLS cell lines. The clinicopathological analyses revealed that the AJCC staging was a significant prognostic factor for overall survival and that the tumor size, depth, and location were significant prognostic factors for event-free survival. Phosphorylated Akt (pAkt), pmTOR, pS6RP, p4E-BP1, pMEK, and pERK expressions were positive in 57.4, 52.4, 71.4, 57.1, 84.1, and 50.8% of the dedifferentiated component of the 63 primary DDLSs. Positive staining for pmTOR was significantly more frequent in the dedifferentiated component than the well-differentiated component. A univariate prognostic analysis revealed that pmTOR expression was associated with poor prognosis in the tumors in the retroperitoneum/ventral body cavity. The mTOR and MEK inhibitors dose-dependently inhibited the cell proliferation of both DDLS cell lines and decreased the expression of downstream pS6RP and pERK, respectively. The combined use of the two inhibitors enhanced antiproliferative activity. In conclusion, the Akt/mTOR and MAPK pathways were activated in DDLS specimens, and the inhibition of these pathways decreased cell proliferation in DDLS cell lines. Our findings suggest that these pathways could be a therapeutic target for patients with DDLS.

MiR-155 Is a Liposarcoma Oncogene That Targets Casein Kinase-1α and Enhances β-Catenin Signaling

Liposarcoma can be an aggressive, debilitating, and fatal malignancy. In this study, we identifed miRNAs associated with the differentiation status of liposarcoma to gain insight into the basis for its progression. miRNA expression profiles determined in human tumors and normal fat specimens identified a dedifferentiated tumor expression signature consisting of 35 miRNAs. Deregulated miRNA expression was confirmed in a second independent sample cohort. The miR-155 was the most overexpressed miRNA and functional investigations assigned an important role in the growth of dedifferentiated liposarcoma cell lines. Transient or stable knockdown of miR-155 retarded tumor cell growth, decreased colony formation, and induced G(1)-S cell-cycle arrest in vitro and blocked tumor growth in murine xenografts in vivo. We identified casein kinase 1α (CK1α) as a direct target of miR-155 control which enhanced β-catenin signaling and cyclin D1 expression, promoting tumor cell growth. In summary, our results point to important functions for miR-155 and β-catenin signaling in progression of liposarcoma, revealing mechanistic vulnerabilities that might be exploited for both prognostic and therapeutic purposes.

Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis

Well-differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n = 84), WDLS (n = 79), and normal fat (n = 23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS when compared to both WDLS and normal fat (15.2- and 27.8-fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBPα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for DDLS.

Establishment and characterization of a novel dedifferentiated liposarcoma cell line, NDDLS‐1

We established a dedifferentiated liposarcoma cell line (NDDLS-1) that produces interleukin-6 (IL-6) and granulocyte-colony stimulating factor (G-CSF). The parental tumor showed high leukemoid reactions. The NDDLS-1 cell line was established from a pleural effusion associated with a lung metastasis. Pleomorphic tumor cells arranged in a haphazard growth pattern were seen in xenograft tumors. Numerous inflammatory cells including neutrophils or eosinophils were present throughout the tumor cells. This finding resembled the dedifferentiated area of the parental tumor. The mice bearing NDDLS-1 showed marked leukocytosis. In addition, the NDDLS-1 cells expressed IL-6 and G-CSF at both the mRNA and protein levels, while the NDDLS-1 cells produced near normal levels of tumor necrosis factor alpha (TNF-α). In the cytogenetic analysis, both the parental tumor and the NDDLS-1 cells showed a ring or giant marker chromosomes. The NDDLS-1 cell line demonstrated the amplification and expression of both MDM2 and CDK4 by fluorescence in situ hybridization and immunohistochemical analysis. The NDDLS-1 cell line is consistent with the parental dedifferentiated liposarcoma, and it should therefore be useful for further investigations of human dedifferentiated liposarcomas.

Abstract 4972: High-throughput functional profiling of dedifferentiated liposarcoma cell lines

Abstract We previously profiled several adult soft-tissue sarcoma subtypes using sequencing, copy number analysis, and gene expression arrays. This high-throughput genetic profiling revealed dozens of candidate genes deserving of further functional validation. To prioritize genes, we performed a pooled shRNA screen to identify essential genes in liposarcoma. Dedifferentiated liposarcoma (DDLS) served as a model for this approach as multiple robust cell lines are available. Five DDLS cell lines (DDLS8817, LPS141, FU-DDLS-1, RDD8107, and LP6) were infected with a pool of 54,020 lentiviral shRNAs targeting ∼11,000 genes (median 5 shRNAs per gene) and passaged for 16 doublings. DNA was extracted and hairpin sequences were PCR amplified and hybridized to a custom microarray that interrogates all shRNAs in the pool. shRNAs were ranked according to their differential abundance between early and late passages. Genes corresponding to the most significantly depleted shRNAs are presumed to affect cell proliferation and are candidate oncogenes. We compared the pattern of hairpin depletion in the DDLS cell lines to a panel of over 60 cell lines representing other common cancers screened with the same pooled shRNA library. The 5 DDLS cell lines clustered into 3 distinct groups. Only one gene appeared to be essential in all 3 groups: WWTR1 (TAZ1), a master-regulator of adipocyte differentiation. Twenty-eight genes appeared essential in at least two DDLS groups including MDM2 and ZBTB2, both reported to negatively regulate the tumor suppressor gene TP53. Low-throughput in vitro experiments have confirmed that WWTR1 knockdown using two shRNA clones inhibits LPS141 proliferation by 78 and 75% after 4 days as compared to scramble (p=0.004 and 0.06 respectively). Our complementary genome-scaled functional screen has confirmed a known oncogene in DDLS and revealed additional candidate genes that appear to have a role in proliferation. Further integration of existing datasets may nominate additional genes essential for liposarcoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4972. doi:10.1158/1538-7445.AM2011-4972

Identification of selective inhibitors of dedifferentiated liposarcoma cells by high-throughput screening (HTS).

10005 Background: Current chemotherapy for dedifferentiated liposarcoma (DDLS) is largely ineffective demanding a need for new therapeutics. An HTS was performed to discover compounds that selectively promote differentiation and cytotoxicity in DDLS cells but not in adipose-derived stem cells (ASC). Methods: A 3,119 molecule HTS was performed using two DDLS cell lines (DDLS8817 and LPS141) and ASC. Cells were incubated with 10uM of compounds and immunofluorescently stained with C/EBPα and Hoechst nuclear stain to quantify differentiation and cytotoxicity. Images were analyzed by laser confocal microscopy. Hits for C/EBPα were validated by immunoblotting. CyQuant proliferation and annexin V staining confirmed cytotoxicity. SCID mice were transplanted with DDLS tumor (BWH) and treated with adriamycin 0.9mg/kg daily or emetine 20mg/kg biweekly. Results: 48 of 3,119 compounds (2%) were positive for C/EBPα induction in both DDLS lines but not in ASC. 32 compounds (1%) were selectively cytotoxic to DDLS lines. 16 compounds, including deguelin and emetine, were selective C/EBPα inducers and cytotoxic. Immunoblotting confirmed increased C/EBPα expression in treated cells (100nM emetine, 4uM deguelin) compared to untreated controls. Emetine completely inhibited proliferation in DDLS lines. Deguelin decreased proliferation by 24% in DDLS8817 (p<0.05) and 48% in LPS141 (p<0.05). ASC treated with deguelin or emetine had no significant change in proliferation. Emetine increased apoptosis 2-fold and 3.4-fold (p<0.05), while deguelin increased apoptosis 1.1-fold and 4.8-fold (p<0.05) compared to controls in DDLS8817 and LPS141. BWH mice treated with emetine had a 49% reduction in tumor growth compared to untreated controls, yet adriamycin only had an 18% reduction. Conclusions: A chemical screen was used to discover compounds that induce differentiation and show selective cytotoxicity for DDLS cells. Emetine inhibits proliferation and induces apoptosis at nM concentrations and inhibits tumor growth in vivo more than adriamycin, a commonly used LS chemotherapeutic. These results show the promise of this approach for the discovery of novel differentiation-based agents for the treatment of DDLS. No significant financial relationships to disclose.

Abstract 3100: The RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 3 is oncogenic in liposarcoma

Abstract Introduction: The insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is an oncofetal mRNA binding protein that controls migration and proliferation through post-transcriptional regulation of target genes. It is aberrantly expressed in various cancers. We sought to determine the alterations in IGF2BP3 expression and copy number in well-differentiated (WD) and dedifferentiated (DD) liposarcoma (LS) and elucidate its role in driving oncogenesis in WD and DD LS cell lines. Methods: U133A microarray analysis was performed on 61 DD and 52 WD LS as well as 17 normal fat (NF) tissue samples. CGH Agilent arrays were used to determine IGF2BP3 amplification status. An shRNA lentiviral system was used to knockdown (KD) IGF2BP3 in DD (DDLS8817, LPS141) and WD (RWD3051) cell lines. Cell proliferation, apoptosis, and mRNA expression were analyzed by Cyquant, annexin V, and qRT-PCR, respectively. Results: CGH analysis showed IGF2BP3 amplification in 19% of DDLS (n=36) and 5% of WDLS (n=36) tumor samples. On U133A analysis IGF2BP3 was 2.5-fold and 1.2-fold upregulated in DDLS and WDLS, respectively, compared to NF. IGF2BP3 was overexpressed in 49% of DDLS and 33% of WDLS tumor samples. Gene expression was increased in DDLS8817 (3-fold), LPS141 (6-fold) and RWD3051 (6-fold) compared to adipose-derived stem cells. IGF2BP3 KD reduced expression &amp;gt;95%(p&amp;lt;0.05) compared to empty vector control for each cell line. IGF2BP3 KD decreased proliferation by 43%, 31%, and 36%(p&amp;lt;0.001) in DDLS8817, LPS141 and RWD3051, respectively, compared to control. Apoptosis was increased by 34%, 55%, and 58%(p&amp;lt;0.0001) compared to control in DDLS8817, LPS141, and RWD3051, respectively, and correlated with level of IGF2BP3 upregulation. p27, a cell cycle inhibitor, was identified as a potential target of IGF2BP3 by photoreactive crosslinking and immunoprecipitation. On Western blot analysis p27 was increased, cyclin D1 was decreased, and p53 was decreased in IGF2BP3 KD compared to control in LPS141, consistent with the functional effects observed. Conclusions: IGF2BP3 overexpression is essential for proliferation and survival of LS cells. The cell cycle regulator, p27 was identified as an IGF2BP3 target mediating these functional effects. Identification of additional IGF2BP3 targets may lead to new therapeutic options for patients with DD and WD liposarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3100.

Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1

MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, small-molecule antagonists of MDM2, the Nutlins, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. However, half of human cancers have mutated p53 and they are resistant to Nutlin treatment. Here, we report that treatment of the p53-mutant malignant peripheral nerve sheath (MPNST) and p53-null HCT116 cells with cisplatin (Cis) and Nutlin-3a induced a degree of apoptosis that was significantly greater than either drug alone. Nutlin-3a also increased the cytotoxicity of both carboplatin and doxorubicin in a series of p53-mutant human tumor cell lines. In the human dedifferentiated liposarcoma cell line (LS141) and the p53 wild-type HCT116 cells, Nutlin-3a induced downregulation of E2F1 and this effect appeared to be proteasome dependent. In contrast, in MPNST and HCTp53-/- cells, Nutlin-3a inhibited the binding of E2F1 to MDM2 and induced transcriptional activation of free E2F1 in the presence of Cis-induced DNA damage. Downregulation of E2F1 by small interfering RNA significantly decreased the level of apoptosis induced by Cis and Nutlin-3a treatment. Moreover, expression of a dominant-negative form of E2F1 rescued cells from apoptosis, whereas cells overexpressing wild-type E2F1 showed an increase in cell death. This correlated with the induction of the proapoptotic proteins p73alpha and Noxa, which are both regulated by E2F1. These results indicate that antagonism of MDM2 by Nutlin-3a in cells with mutant p53 enhances chemosensitivity in an E2F1-dependent manner. Nutlin-3a therefore may provide a therapeutic benefit in tumors with mutant p53 provided it is combined with chemotherapy.

Establishment of a novel human dedifferentiated liposarcoma cell line, FU-DDLS-1: conventional and molecular cytogenetic characterization.

A number of human cell lines derived from well-differentiated, myxoid/round cell, or pleomorphic liposarcoma have been described. To our knowledge, however, no human cell line established from dedifferentiated liposarcoma has been reported. In this study, we established a new human cell line, FU-DDLS-1, which originated from a dedifferentiated liposarcoma arising in the retroperitoneum of a 61-year-old man. This cell line was characterized by immunocytochemistry, conventional banding analysis, fluorescence in situ hybridization with chromosome painting probe, and comparative genomic hybridization (CGH). FU-DDLS-1 cells were spindle or polygonal shaped and possessed oval nuclei and slender cytoplasmic processes. The cultured cells were successfully maintained in vitro for over 90 passages over more than 30 months. The histologic features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original nonlipogenic sarcoma resembling a malignant fibrous histiocytoma. Both in vitro and in vivo, the cells exhibited immunopositive reaction for mdm2 and p53 proteins. Cytogenetically, FU-DDLS-1 displayed a hypertetraploid karyotype with giant marker chromosomes composed partly of chromosome 12 material. In addition, CGH analysis demonstrated that DNA sequence copy number changes including a gain of 12q12-q21 detected in FU-DDLS-1 were essentially the same as those in the original sarcoma. The FU-DDLS-1 cell line, which exhibits the unique conventional and molecular cytogenetic characteristics of dedifferentiated liposarcoma, should be a particularly useful model for studying the molecular pathogenesis of human dedifferentiated liposarcoma.