CXCR4/CXCL12 mediate autocrine cell- cycle progression in NF1-associated malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. They occur sporadically in a subset of patients with neurofibromatosis type 1 (NF1). MPNSTs are highly aggressive, therapeutically resistant, and typically fatal. Using comparative transcriptome analysis, we identified CXCR4, a G-protein-coupled receptor, as highly expressed in mouse models of NF1-deficient MPNSTs, but not in nontransformed precursor cells. The chemokine receptor CXCR4 and its ligand, CXCL12, promote MPNST growth by stimulating cyclin D1 expression and cell-cycle progression through PI3-kinase (PI3K) and ß-catenin signaling. Suppression of CXCR4 activity either by shRNA or pharmacological inhibition decreases MPNST cell growth in culture and inhibits tumorigenesis in allografts and in spontaneous genetic mouse models of MPNST. We further demonstrate conservation of these activated molecular pathways in human MPNSTs. Our findings indicate a role for CXCR4 in NF1-associated MPNST development and identify a therapeutic target.

Experimental models of undifferentiated pleomorphic sarcoma and malignant peripheral nerve sheath tumor.

Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models available, we generated additional cell lines to study these rare cancers. Patient-derived tumors were used to establish 4 new UPS models, including one radiation-associated UPS-UPS271.1, UPS511, UPS0103, and RIS620, one unclassified spindle cell sarcoma-USC060.1, and 3 new models of MPNST-MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing their tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of the cell lines and corresponding xenografts was determined by subcutaneous injection or xenograft re-passaging into immunocompromised mice. USC060.1 and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Targeted sequencing analysis of a panel of genes frequently mutated in sarcomas identified TP53, RB1, and ATRX mutations in a subset of the cell lines. These new cellular models provide the scientific community with powerful tools for detailed studies of tumorigenesis and for investigating novel therapies for UPS and MPNST.

Establishment and characterization of a new human myxoid liposarcoma cell line (DL-221) with the FUS-DDIT3 translocation.

Myxoid liposarcoma has the pathognomonic fusion oncogene FUS-DDIT3 encoding a chimeric transcription factor. Metastatic risk is higher with an increased round cell component and has been linked to aberrations involving the IGFR/PI3K/AKT pathway. These molecular insights have yet to translate to targeted therapies, and the lack of experimental models is a major hindrance. We describe the initial in-depth characterization of a new cell line (DL-221) and establishment of a mouse xenograft model. The cell line DL-221 was derived from a metastatic pleural lesion showing myxoid and round cell histology. This newly established cell line was characterized for phenotypic properties and molecular cytogenetic profile, using PCR, COBRA-FISH, and western blot. Next-generation whole-exome sequencing was performed to further characterize the cell line and the parent tumor. NOD-SCID-IL2R gamma knockout mice were xenograft hosts. DL-221 cells grew an adhering monolayer and COBRA-FISH showed an aneuploid karyotype with t(12;16)(q13;p11) and several other rearrangements; RT-PCR demonstrated a FUS-DDIT3 fusion transcript type 1. Both the cell line and the original tumor harbored a TP53 compound heterozygous mutation in exon 4 and 7, and were wild-type for PIK3CA. Moreover, among the 1254 variants called by whole-exome sequencing, there was 77% concordance between the cell line and parent tumor. The recently described hotspot mutation in the TERT promoter region in myxoid liposarcomas was also found at C228T in DL-221. Xenografts suitable for additional preclinical studies were successfully established in mice after subcutaneous injection. The established DL-221 cell line is the only published available myxoid liposarcoma cell line that underwent spontaneous immortalization, without requiring SV40 transformation. The cell line and its xenograft model are unique and helpful tools to study the biology and novel potential-targeted treatment approaches for myxoid liposarcoma.

The hepatocyte growth factor receptor as a potential therapeutic target for dedifferentiated liposarcoma.

Dedifferentiated liposarcomas (DDLPS) are highly resistant to conventional chemo- and radiotherapies, with surgical resection remaining the classic treatment strategy; therefore, there is a pressing need for novel anti-DDLPS-targeted chemotherapeutics. Hepatocyte growth factor receptor (Met) expression is elevated in DDLPS, but the functional role of Met signaling in this disease is not known. We found that the in vitro stimulation of DDLPS cells with hepatocyte growth factor (HGF) elevated the degree of PI3K/AKT and MAPK pathway signaling, and that pro-tumorigenic phenotypes such as cell proliferation, invasion, and migration were significantly enhanced. Conversely, Met knockdown using shRNA-mediated interference decreased HGF-induced Met signaling, the invasive and migratory nature of DDLPS cells in vitro, and the tumorigenicity of DDLPS cells in vivo. These data strongly support the role for Met as a DDLPS therapeutic target. To that end, using EMD1214063, an ATP-competitive kinase inhibitor that targets Met more specifically than other kinases, inhibited Met-dependent signaling, reduced the oncogenicity of DDLPS cells in vitro, and significantly increased the survival of nude mice bearing subcutaneous DDLPS xenografts. These findings support further investigations of HGF-induced Met signaling inhibition in DDLPS, as a potential strategy to enhance clinical outcomes for this disease.

Targeting the Notch pathway: A potential therapeutic approach for desmoid tumors.

BACKGROUND: Desmoid tumors (DTs) are rare mesenchymal lesions that can recur repeatedly. When it is feasible, DTs are surgically resected; however, this often results in high recurrence rates. Recently, treatment with PF-03084014, a potent γ-secretase inhibitor, has been shown to have antitumor activity in several tumor types by affecting the WNT/ß-catenin pathway. Consequently, Notch pathway inhibition by PF-03084014 might be a promising approach for DT treatment. METHODS: The expression of Notch pathway components was analyzed in DT tissues and cell strains with immunohistochemistry and Western blotting, respectively. A panel of DT cell strains was exposed to PF-03084014 and evaluated for cell proliferation. Antitumor effects were assessed via cell cycle, apoptosis, and migration and invasion analysis. Cells treated with PF-03084014 were characterized with a gene array analysis combined with Ingenuity Pathway Analysis. RESULTS: The results showed that Notch pathway components were expressed at different levels in DTs. Hes1 (Hes Family BHLH Transcription Factor 1) was overexpressed in DT tumors versus dermal scar tissue, and PF-03084014 caused significant decreases in Notch intracellular domain and Hes1 expression in DT cell strains. PF-03084014 decreased DT cell migration and invasion and also caused cell growth inhibition in DT cell strains, most likely through cell cycle arrest. Gene array analysis combined with Ingenuity Pathway Analysis showed that Wnt1-inducible signaling pathway protein 2 possibly regulated Notch and WNT pathways after treatment with PF-03084014 through integrin. CONCLUSION: Our findings suggest that the Notch pathway is an important DT therapeutic target. Furthermore, PF-03084014 has significant antitumor activity against DTs, and it may be an alternative strategy for DT treatment.

AXL is a potential therapeutic target in dedifferentiated and pleomorphic liposarcomas.

BACKGROUND: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined. METHODS: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo. RESULTS: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo. CONCLUSIONS: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.

Involvement of the PI3K/Akt pathway in myxoid/round cell liposarcoma.

The molecular determinants involved in the progression of myxoid liposarcoma to increased cellularity/round cell change are poorly understood. We studied the PI3K/Akt pathway in myxoid and round cell liposarcomas using a tissue microarray composed of 165 tumors from 111 patients, and mutational analysis of PIK3CA in 44 cases. Activating PIK3CA mutations were found in 6/44 cases, 14%; mutations were more frequent in round cell vs myxoid tumors (5/15, 33% vs 1/29, 3%; P=0.013). Complete loss of PTEN, an alternative mechanism for PI3K/Akt activation, was found in 13/111 (12%) cases and was mutually exclusive with PIK3CA mutation. Strong IGF1R expression was demonstrated in 14/39 (36%) of round cell and 11/58 (19%) of myxoid tumors (P=0.062). Activation of the PI3K pathway was confirmed using immunohistochemical analysis for downstream targets phospho-S6 ribosomal protein and phospho-4EBP1. Phospho-4EBP1 was increased in round cell tumors compared with myxoid tumors (24/30, 80% vs 25/44, 57%; P=0.038) or tumors with treatment effect (10/24, 42%; P=0.02). Phospho-S6 was highly expressed in both myxoid and round cell tumors (29/47, 62% and 14/30, 47%, respectively; P=0.2). In tumors with PIK3CA mutation, any IGF1R expression, or loss of PTEN expression, phospho-4EBP1 was more frequently elevated compared with tumors without a known activating event in the PI3K pathway (55/72; 76% vs 3/8, 38%; P=0.033). These findings suggest that activation of the PI3K/Akt pathway via activating mutation of PIK3CA, loss of PTEN, or IGF1R expression have a role in round cell transformation. The PI3K/Akt pathway may therefore provide a therapeutic target in round cell liposarcoma.

Increased midkine expression correlates with desmoid tumour recurrence: a potential biomarker and therapeutic target.

Desmoid tumours (DTs) are soft tissue monoclonal neoplasms exhibiting a unique phenotype, consisting of aggressive local invasiveness without metastatic capacity. While DTs can infrequently occur as part of familial adenomatosis polyposis, most cases arise sporadically. Sporadic DTs harbour a high prevalence of CTNNB1 mutations and hence increased ß-catenin signalling. However, ß-catenin downstream transcriptional targets and other molecular deregulations operative in DT inception and progression are currently not well defined, contributing to the lack of sensitive molecular prognosticators and efficacious targeted therapeutic strategies. We compared the gene expression profiles of 14 sporadic DTs to those of five corresponding normal tissues and six solitary fibrous tumour specimens. A DT expression signature consisting of 636 up- and 119 down-regulated genes highly enriched for extracellular matrix, cell adhesion and wound healing-related proteins was generated. Furthermore, 98 (15%) of the over-expressed genes were demonstrated to contain a TCF/LEF consensus binding site in their promoters, possibly heralding direct ß-catenin downstream targets relevant to DT. The protein products of three of the up-regulated DT genes: ADAM12, MMP2 and midkine, were found to be commonly expressed in a large cohort of human DT samples assembled on a tissue microarray. Interestingly, enhanced midkine expression significantly correlated with a higher propensity and decreased time for primary DT recurrence (log-rank p = 0.0025). Finally, midkine was found to enhance the migration and invasion of primary DT cell cultures. Taken together, these studies provide insights into potential DT molecular aberrations and novel ß-catenin transcriptional targets. Further studies to confirm the utility of midkine as a clinical DT molecular prognosticator and a potential therapeutic target are therefore warranted. Raw gene array data can be found at: http://smd.stanford.edu/

An experimental model for the study of well-differentiated and dedifferentiated liposarcoma; deregulation of targetable tyrosine kinase receptors.

Therapeutic progress in well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS) is hampered by lack of relevant experimental models, thereby limiting comprehensive molecularly based investigations. Our goal is to bridge this experimental gap by establishing and characterizing an in vitro/in vivo model useful for examining WDLPS/DDLPS molecular pathogenesis and also therapeutic screening and testing. WDLPS/DDLPS cells were isolated from freshly resected human surgical specimens and were phenotypically and molecularly characterized. MDM2 amplification was determined via FISH analysis. Adipogenic differentiation was evaluated using Oil Red O staining and western blotting (WB). Tyrosine kinase receptors' (TKRs) expression in pre-adipocytes, adipocytes, WDLPS, and DDLPS cells was determined via western blot analysis. SCID mouse xenograft growth was assessed after subcutaneous and/or intraperitoneal tumor cell injection. There was enhanced proliferation, migration, invasion, survival, and pro-angiogenic capacity in DDLPS cells vs WDLPS cells. DDLPS cells formed tumors in SCID mice whereas WDLPS did not. WDLPS/DDLPS cells, especially those that exhibited baseline PPARγ expression, partially retained terminal adipogenic differentiation capacity. MDM2 amplification was found in all WDLPS/DDLPS cell strains, CDK4 overexpression was observed in LPS cells as compared with normal adipocytes, and enhanced JUN expression and phosphorylation was seen in DDLPS cells as compared with WDLPS cells. The TKRs: MET, AXL, KIT, and IGF-1R were overexpressed in LPS cells vs normal adipocytes and pre-adipocytes. In conclusion, these newly established cellular and xenograft models can facilitate investigation of liposarcomagenesis, dedifferentiation, and tumor progression. Further studies of the molecular deregulations so identified may lead to improved therapeutic strategies for patients afflicted by these unfavorable malignancies.

Pleomorphic liposarcoma: clinical observations and molecular variables.

BACKGROUND: Pleomorphic liposarcoma (PLS) is a rare high-grade sarcoma that has lipoblastic differentiation. In this study, the authors evaluated PLS natural history, patient outcomes, and commonly deregulated protein biomarkers. METHODS: Medical records from patients (n = 155) who had PLS from 1993 to 2010 were reviewed. Univariate and multivariate analyses were conducted to identify independent prognosticators. A PLS tissue microarray (TMA) (n = 56 patient specimens) was constructed for immunohistochemical analysis of molecular markers, and p53 gene sequencing (exons 5-9) was conducted. RESULTS: The average patient age was 57 years, and the patients presented with primary disease (n = 102), recurrent disease (n = 16), and metastatic disease (n = 37). Lower extremity was the most common disease site (40%), and the average tumor size was 11 cm. Complete follow-up data were available for 83 patients, and their median follow-up was 22.6 months. The 5-year disease-specific survival rate was 53%; and recurrent disease, unresectability, and microscopic positive margins were identified as predictors of a poor prognosis. Systemic relapse (the strongest poor prognostic determinant) developed in 35% of patients with localized PLS. Immunohistochemical analysis revealed increased expression of peroxisome proliferator-activated receptor gamma (an adipogenic marker), B-cell leukemia 2 and survivin (survival factors), vascular endothelial growth factor (an angiogenic factor), matrix metalloproteinase 2, and other biomarkers. Frequent loss of retinoblastoma protein expression and high p53 mutation rates (approximately 60%) were observed. CONCLUSIONS: PLS is an aggressive, metastasizing sarcoma. Identifying ubiquitous molecular events underlying PLS progression is crucial for progress in patient management and outcomes.

Epithelioid sarcoma and unclassified sarcoma with epithelioid features: clinicopathological variables, molecular markers, and a new experimental model.

BACKGROUND: Epithelioid sarcoma (ES) and unclassified sarcoma with epithelioid features (USEF) are clinically and therapeutically unresolved. We compared ES and USEF patients' clinical behavior, treatment, outcome, and molecular marker expression. Furthermore, preclinical ES study models were developed to enable comprehensive benchside investigations. PATIENTS AND METHODS: A database of ES and USEF patients (n = 116) treated since 1992 was created. A clinically annotated ES-USEF tissue microarray (TMA) was assayed for tumor-related markers. Newly established human and commercially available ES cell lines were characterized and tested in vivo. RESULTS: ES and USEF patients presenting with localized disease exhibited 22% and 25% local recurrence rates, 35% and 19% nodal metastasis rates, and 41% and 53% distant metastasis rates (median follow-up, 54 months and 39 months, respectively). The 5- and 10-year disease-specific survival rates were 88% and 43% and 52% and 42% (ES and USEF, respectively). TMA immunohistochemistry identified integrase interactor (INI)-1 loss, cancer antigen 125, and p53 nuclear expression as significantly more common in ES than USEF cases. Both cell lines preserved ES morphological and biochemical characteristics in vitro and in vivo; loss of INI-1 was shown to occur in both lines. CONCLUSIONS: Enhanced knowledge of ES and USEF clinical behavior, marker expression, and molecular determinants, extended via experimental models, will hopefully accelerate development of urgently needed effective targeted therapies for ES and USEF.

'Difficult to diagnose' desmoid tumours: a potential role for CTNNB1 mutational analysis.

AIMS: The utility of CTNNB1 (encoding ß-catenin) genotyping for diagnosing sporadic desmoid tumours (DT) when traditional clinicopathological parameters were inconclusive was evaluated. METHODS AND RESULTS: Cases included were: (i) new primary lesions where initial DT diagnosis was inconclusive; and (ii) possible recurrent DT versus scar. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained via needle biopsy or a surgical excision (57 specimens) as part of initial assessment. DNA extraction, CTNNB1 exon 3 amplification and sequencing were conducted in a Clinical Laboratory Improvement Amendments of 1988 (CLIA)-approved molecular diagnostics laboratory. For patients with no previous DT history (n = 47) sequencing identified mutations in 30 (64%), substantiating DT diagnosis. In biopsies with non-mutated (NM) CTNNB1 (n = 17) the test was inconclusive; in seven of these, a diagnosis of DT was strongly favoured in the subsequent surgical resection specimen. Ten patients with previously resected DT were evaluated; mutation was identified in six cases (60%), indicating DT over scar. In two (20%) with primary tumours harbouring CTNNB1 mutation no mutation was found, favouring scar over DT; the other two NM-CTNNB1 cases (20%) were inconclusive. CONCLUSIONS: CTNNB1 genotyping can be very useful in 'difficult to diagnose' lesions when the differential diagnosis includes DT. Recognizing inherent test limitations, the presence of CTNNB1 mutation can inform the therapeutic approach.

Specific mutations in the beta-catenin gene (CTNNB1) correlate with local recurrence in sporadic desmoid tumors.

Desmoid fibromatosis is a rare, nonmetastatic neoplasm marked by local invasiveness and relentless recurrence. Molecular determinants of desmoid recurrence remain obscure. beta-Catenin deregulation has been commonly identified in sporadic desmoids although the incidence of CTNNB1 (the gene encoding beta-catenin) mutations is uncertain. Consequently, we evaluated the prevalence of CTNNB1 mutations in a large cohort of sporadic desmoids and examined whether mutation type was relevant to desmoid outcome. Desmoid specimens (195 tumors from 160 patients, 1985 to 2005) and control dermal scars were assembled into a clinical data-linked tissue microarray. CTNNB1 genotyping was performed on a 138-sporadic desmoid subset. Immunohistochemical scoring was performed per standard criteria and data were analyzed using Kaplan-Meier and other indicated methods. CTNNB1 mutations were observed in 117 of 138 (85%) of desmoids. Three discrete mutations in two codons of CTNNB1 exon 3 were identified: 41A (59%), 45F (33%), and 45P (8%, excluded from further analysis because of rarity). Five-year recurrence-free survival was significantly poorer in 45F-mutated desmoids (23%, P < 0.0001) versus either 41A (57%) or nonmutated tumors (65%). Nuclear beta-catenin expression was observed in 98% of specimens and intensity was inversely correlated with incidence of desmoid recurrence (P < 0.01). In conclusion, CTNNB1 mutations are highly common in desmoid tumors. Furthermore, patients harboring CTNNB1 (45F) mutations are at particular risk for recurrence and therefore may especially benefit from adjuvant therapeutic approaches.

Combined vascular endothelial growth factor receptor/epidermal growth factor receptor blockade with chemotherapy for treatment of local, uterine, and metastatic soft tissue sarcoma.

PURPOSE: Soft tissue sarcoma (STS) is a rare heterogeneous malignancy. Overall survival has been stagnant for decades, primarily because systemic therapies are ineffective versus metastases, the leading cause of STS lethality. Consequently, we examined whether tyrosine kinase receptors active in STS growth signaling might be blockable and whether multireceptor blockade might synergize with low-dose STS chemotherapy by therapeutically affecting STS cells and their associated microenvironment. EXPERIMENTAL DESIGN: Vandetanib (AstraZenca), a tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 and epidermal growth factor receptor, was evaluated alone and with chemotherapy in vitro and in vivo in three human STS nude mouse xenograft models of different STS locations (muscle, uterus, lung), stages (primary, metastatic), and subtypes (leiomyosarcoma, fibrosarcoma, uterine sarcoma: luciferase-expressing MES-SA human uterine sarcoma cells surgically implanted into uterine muscularis with bioluminescence tumor growth assessment; developed by us). RESULTS: In vitro, human STS cells were sensitive to vandetanib. Vandetanib alone and with chemotherapy statistically significantly inhibited leiomyosarcoma local growth and fibrosarcoma lung metastasis. Direct injection of MES-SA into nude mice uterine muscularis resulted in high tumor take (88%), whereas s.c. injection resulted in no growth, suggesting microenvironmental tumor growth modulation. Vandetanib alone and with chemotherapy statistically significantly inhibited uterine sarcoma growth. In all models, vandetanib induced increased apoptosis, decreased tumor cell proliferation, and decreased angiogenesis. CONCLUSIONS: Vandetanib has antitumor effects against human STS subtypes in vitro and in vivo, where it also affects the tumor-associated microenvironment. Given the urgent need for better systemic approaches to STS, clinical trials evaluating vandetanib, perhaps with low-dose chemotherapy, seem warranted.

Rad51 overexpression contributes to chemoresistance in human soft tissue sarcoma cells: a role for p53/activator protein 2 transcriptional regulation.

We investigated whether Rad51 overexpression plays a role in soft tissue sarcoma (STS) chemoresistance as well as the regulatory mechanisms underlying its expression. The studies reported here show that Rad51 protein is overexpressed in a large panel of human STS specimens. Human STS cell lines showed increased Rad51 protein expression, as was also observed in nude rat STS xenografts. STS cells treated with doxorubicin exhibited up-regulation of Rad51 protein while arrested in the S-G(2) phase of the cell cycle. Treatment with anti-Rad51 small interfering RNA decreased Rad51 protein expression and increased chemosensitivity to doxorubicin. Because we previously showed that reintroduction of wild-type p53 (wtp53) into STS cells harboring a p53 mutation led to increased doxorubicin chemosensitivity, we hypothesized that p53 participates in regulating Rad51 expression in STS. Reintroduction of wtp53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression. Using luciferase reporter assays, we showed that reconstitution of wtp53 function decreased Rad51 promoter activity. Deletion constructs identified a specific Rad51 promoter region containing a p53-responsive element but no p53 consensus binding site. Electrophoretic mobility shift assays verified activator protein 2 (AP2) binding to this region and increased AP2 binding to the promoter in the presence of wtp53. Mutating this AP2 binding site eliminated the wtp53 repressive effect. Furthermore, AP2 knockdown resulted in increased Rad51 expression. In light of the importance of Rad51 in modulating STS chemoresistance, these findings point to a potential novel strategy for molecular-based treatments that may be of relevance to patients burdened by STS.

Wild-type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis.

Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis-defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-kappaB (NF-kappaB) site. Mutating this NF-kappaB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-kappaB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation.

Co-targeting PI3K, mTOR, and IGF1R with small molecule inhibitors for treating undifferentiated pleomorphic sarcoma.

Undifferentiated pleomorphic sarcomas (UPSs) are aggressive mesenchymal malignancies with no definitive cell of origin or specific recurrent genetic hallmarks. These tumors are largely chemoresistant; thus, identification of potential therapeutic targets is necessary to improve patient outcome. Previous studies demonstrated that high expression of activated protein kinase B (AKT) in patients with UPS corresponds to poor disease-specific survival. Here, we demonstrate that inhibiting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling using a small molecule inhibitor reduced UPS cell proliferation and motility and xenograft growth; however, increased phosphorylation of insulin-like growth factor 1 receptor (IGF1R) indicated the potential for adaptive resistance following treatment through compensatory receptor activation. Co-treatment with a dual PI3K/mTOR inhibitor and an anti-IGF1R kinase inhibitor reduced in vivo tumor growth rates despite a lack of antiproliferative effects in vitro. Moreover, this combination treatment significantly decreased UPS cell migration and invasion, which is linked to changes in p27 subcellular localization. Our results demonstrate that targeted inhibition of multiple components of the IGF1R/PI3K/mTOR pathway was more efficacious than single-agent therapy and suggest that co-targeting this pathway could be a beneficial therapeutic strategy for patients with UPS.

HDAC Inhibition for the Treatment of Epithelioid Sarcoma: Novel Cross Talk Between Epigenetic Components.

UNLABELLED: Epithelioid sarcoma is a rare neoplasm uniquely comprised of cells exhibiting both mesenchymal and epithelial features. Having propensity for local and distant recurrence, it poses a diagnostic dilemma secondary to pathologic complexity. Patients have dismal prognosis due to lack of effective therapy. HDAC inhibitors (HDACi) exhibit marked antitumor effects in various malignancies. The studies here demonstrate that pan-HDAC inhibitors constitute novel therapeutics versus epithelioid sarcoma. Human ES cells (VAESBJ, HS-ES, Epi-544) were studied in preclinical models to evaluate HDACi effects. Immunoblot and RT-PCR were used to evaluate expression of acetylated tubulin, histones H3/H4, EZH2 upon HDACi. MTS and clonogenic assays were used to assess the impact of HDACi on cell growth. Cell culture assays were used to evaluate the impact of HDACi and EZH2-specific siRNA inhibition on cell-cycle progression and survival. Unbiased gene array analysis was used to identify the impact of HDACi on epithelioid sarcoma gene expression. Xenografts were used to evaluate epithelioid sarcoma tumor growth in response to HDACi. HDAC inhibition increased target protein acetylation and abrogated cell growth and colony formation in epithelioid sarcoma cells. HDACi induced G(2) cell-cycle arrest and marked apoptosis, and reduced tumor growth in xenograft models. HDACi induced widespread gene expression changes, and EZH2 was significantly downregulated. EZH2 knockdown resulted in abrogated cell growth in vitro. IMPLICATIONS: The current study suggests a clinical role for HDACi in human epithelioid sarcoma, which, when combined with EZH2 inhibitors, could serve as a novel therapeutic strategy for epithelioid sarcoma patients. Future investigations targeting specific HDAC isoforms along with EZH2 may potentially maximizing treatment efficacy.

SAR405838: A Novel and Potent Inhibitor of the MDM2:p53 Axis for the Treatment of Dedifferentiated Liposarcoma.

PURPOSE: Dedifferentiated liposarcoma (DDLPS) is an aggressive malignancy that can recur locally or disseminate even after multidisciplinary care. Genetically amplified and expressed MDM2, often referred to as a "hallmark" of DDLPS, mostly sustains a wild-type p53 genotype, substantiating the MDM2:p53 axis as a potential therapeutic target for DDLPS. Here, we report on the preclinical effects of SAR405838, a novel and highly selective MDM2 small-molecule inhibitor, in both in vitro and in vivo DDLPS models. EXPERIMENTAL DESIGN: The therapeutic effectiveness of SAR405838 was compared with the known MDM2 antagonists Nutlin-3a and MI-219. The effects of MDM2 inhibition were assessed in both in vitro and in vivo. In vitro and in vivo microarray analyses were performed to assess differentially expressed genes induced by SAR405838, as well as the pathways that these modulated genes enriched. RESULTS: SAR405838 effectively stabilized p53 and activated the p53 pathway, resulting in abrogated cellular proliferation, cell-cycle arrest, and apoptosis. Similar results were observed with Nutlin-3a and MI-219; however, significantly higher concentrations were required. In vitro effectiveness of SAR405838 activity was recapitulated in DDLPS xenograft models where significant decreases in tumorigenicity were observed. Microarray analyses revealed genes enriching the p53 signaling pathway as well as genomic stability and DNA damage following SAR405838 treatment. CONCLUSIONS: SAR405838 is currently in early-phase clinical trials for a number of malignancies, including sarcoma, and our in vitro and in vivo results support its use as a potential therapeutic strategy for the treatment of DDLPS.