Liposarcoma Cell Lines Research Articles

Abstract B09: Novel sarcoma treatments identified via drug sensitivity/resistance screening of sarcoma cell lines

Abstract Sarcomas comprise a plethora of subtypes and each should be regarded as ultra-orphan malignancy. Over the last century incremental improvements of sarcoma therapies have provided better survival; however, treatment options appear to have been exhausted and new alternatives beyond traditional chemotherapeutic regimens are much needed. To broaden the repertoire of antisarcoma drugs, we applied a direct drug testing approach. Using a library of 349 anticancer compounds (FDA-approved/under clinical trial) and panels of liposarcoma and osteosarcoma cell lines, we selected a group of potential novel antisarcoma therapeutics and retested them against cells from primary tumors. We are also investigating genomic and molecular features to find potential drug response biomarkers. Since all those compounds have been approved or are under clinical trials, they present a unique shortcut repurposing opportunity for personalized sarcoma treatment. Citation Format: Iwona Grad, Pilar Ayuda, Robert Hanes, Jorrit Enserink, Ola Myklebost. Novel sarcoma treatments identified via drug sensitivity/resistance screening of sarcoma cell lines [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B09.

Microarray, IPA and GSEA Analysis in Mice Models.

This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mdx mouse model and to analyze previously published microarray data of human liposarcoma cell lines.

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

BackgroundLiposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management.MethodsLarge RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial.ResultsScreening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model.ConclusionsTwo large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Tumor Suppression Efficacy of Heat Shock Protein 90 Inhibitor 17AAG in a Liposarcoma Mouse Model.

Heat shock protein 90 (HSP90) inhibitors have recently been tested as anticancer drugs in a variety of carcinomas. Yet, there exist only few reports about HSP90 inhibitor and its thepeutic effect on liposarcoma. The therapeutic effects of HSP90 inhibitors have been mainly observed in oncogenic and tumor angiogenic signaling cascades by observing tumor growth. We used the the LPS 246 liposarcoma cell line and GS-076 PDC (patient-derived cell lines). On these, we performed cell viability assays and migration assays under treatment with the HSP90 inhibitor, 17AAG. For analyzing angiogenesis factor, we used quantitative polymerase chain reaction (qPCR) after treating cells with the 17AAG inhibitor. Regarding in vivo assay, we made the tumor model in immune-deficient mouse and compared the tumor size of drug-treated group at each time point with controls. For sequestering analysis of angiogenesis factor in vivo, we performed immuno-fluorescence (IF) staining on tumor tissue. Through cell viability, migration assay and qPCR about angiogenesis factor, we demonstrated the anti-oncogenic and anti-angiogenic effects of an HSP90 inhibitor on a liposarcoma cell line and a patient-derived primary cell model (PDC). Also, the HSP90 inhibitor 17AAG effectively inhibited the activity of protein kinase B (AKT) and blocked extracellular signal-regulated kinase (ERK) activity. Hence, 17AAG effectively disrupted the oncogenic signaling cascade and substantially inhibited tumor growth in vitro. In an LPS 863 cell xenograft mouse model treated with 17AAG, we observed that tumor size was decreasing, as well as down-regulation of the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and signal transducer and activator of transcription-3 (STAT3). 17AAG reduced the activity of AKT, ERK, VEGF and STAT3 in oncogenic and angiogenic pathways in liposarcoma PDC models derived from patients' tissues and cancer cell lines.

FUS–DDIT3 Fusion Protein-Driven IGF-IR Signaling is a Therapeutic Target in Myxoid Liposarcoma

Purpose: Myxoid liposarcoma is an aggressive disease with particular propensity to develop hematogenic metastases. Over 90% of myxoid liposarcoma are characterized by a reciprocal t(12;16)(q13;p11) translocation. The resulting chimeric FUS-DDIT3 fusion protein plays a crucial role in myxoid liposarcoma pathogenesis; however, its specific impact on oncogenic signaling pathways remains to be substantiated. We here investigate the functional role of FUS-DDIT3 in IGF-IR/PI3K/Akt signaling driving myxoid liposarcoma pathogenesis.Experimental Design: Immunohistochemical evaluation of key effectors of the IGF-IR/PI3K/Akt signaling axis was performed in a comprehensive cohort of myxoid liposarcoma specimens. FUS-DDIT3 dependency and biological function of the IGF-IR/PI3K/Akt signaling cascade were analyzed using a HT1080 fibrosarcoma-based myxoid liposarcoma tumor model and multiple tumor-derived myxoid liposarcoma cell lines. An established myxoid liposarcoma avian chorioallantoic membrane model was used for in vivo confirmation of the preclinical in vitro results.Results: A comprehensive subset of myxoid liposarcoma specimens showed elevated expression and phosphorylation levels of various IGF-IR/PI3K/Akt signaling effectors. In HT1080 fibrosarcoma cells, overexpression of FUS-DDIT3 induced aberrant IGF-IR/PI3K/Akt pathway activity, which was dependent on transcriptional induction of the IGF2 gene. Conversely, RNAi-mediated FUS-DDIT3 knockdown in myxoid liposarcoma cells led to an inactivation of IGF-IR/PI3K/Akt signaling associated with diminished IGF2 mRNA expression. Treatment of myxoid liposarcoma cell lines with several IGF-IR inhibitors resulted in significant growth inhibition in vitro and in vivoConclusions: Our preclinical study substantiates the fundamental role of the IGF-IR/PI3K/Akt signaling pathway in myxoid liposarcoma pathogenesis and provides a mechanism-based rationale for molecular- targeted approaches in myxoid liposarcoma cancer therapy. Clin Cancer Res; 23(20); 6227-38. ©2017 AACR.

Abstract 4188: Multitargeted tyrosine kinase inhibitor identified as potential therapeutic intervention for liposarcoma using high-throughput profiling

Abstract Liposarcoma is a rare fat cell adult tumor with high risk of recurrence and metastasis, largely underserved by research community and till now limited progress has been made in treatment of this aggressive disease. We used a strategy to identify effective and potential therapeutic kinase inhibitors, irrespective of the activated kinase pathway, using small molecule kinase inhibitor panel. We screened liposarcoma cell lines of different histotypes to a panel of small molecule kinase inhibitors and analyzed using cell proliferation assay. In vitro cell proliferation assay, colony formation, cell cycle analysis, apoptosis assay and western blotting analysis were performed to investigate the effect and mechanism of inhibitor treatment on liposarcoma. Liposarcoma xenograft model system was also employed to investigate anti-tumor in vivo effects of inhibitor treatment. We observed that cell lines demonstrated diverse in vitro drug sensitivity patterns to various kinase inhibitors. Most of the cell lines showed very high sensitivity towards inhibitors targeting proteasome, protein kinase C, Hsp90, PI3K, mTOR and CDKs. Among the receptor tyrosine kinase inhibitors; Ponatinib, Dasatinib, and Sunitinib are the top most sensitive drugs affecting liposarcoma cells lines’ viability irrespective of subtypes and all these are already approved by the U.S. Food and Drug Administration to treat various cancers. We studied multi-targeted tyrosine kinase inhibitor ponatinib as an effective potential drug molecule in liposarcoma. We demonstrated that ponatinib treatment in liposarcoma cell lines reduced the levels of phosphorylated KIT compared to total KIT protein levels in dose dependent manner. Further, western blotting experiments revealed effect of ponatinib treatment on KIT downstream signalling by inhibiting the phosphorylation of AKT, ERK1/2, STAT3, mTOR, P70S6K and RB without affecting their total protein levels. Significant reduction in cell number and colonies with ponatinib treatment implicates anti-neoplastic effects in liposarcoma. Ponatinib treatment causes cell cycle arrest at G0/G1 phase by regulating CDK4 and cyclinD1 proteins levels. It also induces apoptosis of treated cells by downregulating AKT and ERK signaling pathway leading to dephosphorylation of BAD. Similar growth inhibiting effect of ponatinib was demonstrated in in vivo liposarcoma xenograft model. In vitro drug sensitivity profiling of liposarcoma highlighted Ponatinib, an oral FDA approved drug, as potential therapeutic drug candidate for treatment and management of this deadly tumor. Citation Format: Deepika Kanojia, Manoj Garg, Jakki Martinez, Anand M.T., Samuel B Luty, Ngan B Doan, Jonathan W Said, Jeffrey W Tyner, H Phillip Koeffler. Multitargeted tyrosine kinase inhibitor identified as potential therapeutic intervention for liposarcoma using high-throughput profiling [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 4188. doi:10.1158/1538-7445.AM2017-4188

TP53INP2-related basal autophagy is involved in the growth and malignant progression in human liposarcoma cells.

Understanding the function of autophagy may allow us to develop a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes in the treatment of cancers. Here, we studied the contribution of basal autophagy in human liposarcoma. The levels of basal autophagy were analyzed by measuring autophagy-related protein expression and autophagosome formation. TP53INP2 expression was determined by real-time PCR, western blot and tissue microarray. Genetic inhibition or overexpression of TP53INP2 was performed to examine its effects on autophagic activity and cell growth. Compared with human liposarcoma cell line SW872, low level of basal autophagy were present in SW872-S cells with high malignancy. Moreover, a decrease of TP53INP2 expression was found accompanying liposarcoma malignant progression in cell lines and primary tissues. TP53INP2 expression was required for autophagic activity in liposarcoma cells. Autophagy inhibition with chloroquine suppressed the growth of liposarcoma cells. TP53INP2-related basal autophagy rendered liposarcoma cells to be more resistant to bortezomib-induced inhibition of cell growth. The results reveal the association of TP53INP2-related basal autophagy with cell growth and malignant progression of human liposarcoma, which helps re-evaluate targeting autophagy for cancer therapy, and suggest that TP53INP2 expression might be used as a prognostic marker to predict human liposarcoma malignancies.

Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays

ABSTRACTBackground: The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Methods: Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). Results: As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Conclusion: Curcumin and VAE can inhibit the proliferation and viability of STS cells.

Anti‑proliferative activity of epigallocatechin‑3‑gallate and silibinin on soft tissue sarcoma cells.

Disseminated soft tissue sarcomas (STS) present a therapeutic dilemma. The first-line cytostatic doxorubicin demonstrates a response rate of 30% and is not suitable for elderly patients with underlying cardiac disease, due to its cardiotoxicity. Well-tolerated alternative treatment options, particularly in palliative situations, are rare. Therefore, the present study assessed the anti-proliferative effects of the natural compounds epigallocatechin-3-gallate (EGCG), silibinin and noscapine on STS cells. A total of eight different human STS cell lines were used in the study: Fibrosarcoma (HT1080), liposarcoma (SW872, T778 and MLS-402), synovial sarcoma (SW982, SYO1 and 1273) and pleomorphic sarcoma (U2197). Cell proliferation and viability were analysed by 5-bromo-2′-deoxyuridine and MTT assays and real-time cell analysis (RTCA). RTCA indicated that noscapine did not exhibit any inhibitory effects. By contrast, EGCG decreased proliferation and viability of all cell lines except for the 1273 synovial sarcoma cell line. Silibinin exhibited anti-proliferative effects on all synovial sarcoma, liposarcoma and fibrosarcoma cell lines. Liposarcoma cell lines responded particularly well to EGCG while synovial sarcoma cell lines were more sensitive to silibinin. In conclusion, the green tea polyphenol EGCG and the natural flavonoid silibinin from milk thistle suppressed the proliferation and viability of liposarcoma, synovial sarcoma and fibrosarcoma cells. These compounds are therefore potential candidates as mild therapeutic options for patients that are not suitable for doxorubicin-based chemotherapy and require palliative treatment. The findings from the present study provide evidence to support in vivo trials assessing the effect of these natural compounds on solid sarcomas.

Molecular mechanism and therapeutic implications of selinexor (KPT-330) in liposarcoma.

Exportin-1 mediates nuclear export of multiple tumor suppressor and growth regulatory proteins. Aberrant expression of exportin-1 is noted in human malignancies, resulting in cytoplasmic mislocalization of its target proteins. We investigated the efficacy of selinexor against liposarcoma cells both in vitro and in vivo. Exportin-1 was highly expressed in liposarcoma samples and cell lines as determined by immunohistochemistry, western blot, and immunofluorescence assay. Knockdown of endogenous exportin-1 inhibited proliferation of liposarcoma cells. Selinexor also significantly decreased cell proliferation as well as induced cell cycle arrest and apoptosis of liposarcoma cells. The drug also significantly decreased tumor volumes and weights of liposarcoma xenografts. Importantly, selinexor inhibited insulin-like growth factor 1 (IGF1) activation of IGF-1R/AKT pathway through upregulation of insulin-like growth factor binding protein 5 (IGFBP5). Further, overexpression and knockdown experiments showed that IGFBP5 acts as a tumor suppressor and its expression was restored upon selinexor treatment of liposarcoma cells. Selinexor decreased aurora kinase A and B levels in these cells and inhibitors of these kinases suppressed the growth of the liposarcoma cells. Overall, our study showed that selinexor treatment restored tumor suppressive function of IGFBP5 and inhibited aurora kinase A and B in liposarcoma cells supporting the usefulness of selinexor as a potential therapeutic strategy for the treatment of this cancer.

Abstract IA04: The Targeting of CDK4/6: Have we gone full circle?

Abstract Cyclin Dependent kinase 4 (CDK4) is a critical kinase that regulates the G1/S phase transition within the cell cycle. There has been a major effort to develop drugs that inhibit this kinase. The first CDK4 inhibitor to be approved is Palbociclib. It is approved in combination with letrozole for the treatment of postmenopausal women with metastatic estrogen receptor (ER)-positive breast cancer as initial endocrine-based therapy. In this study Palbociclib (Palbo) improved median progression free survival (mPFS) from 10.2 months with letrozole alone to 20.2 months with Palbo in the combination therapy [Hazard Ratio (HR) 0.488 (95% CI 0.319, 0.748)]. Response rates were 55.4% with the combination and 39.4% with letrozole alone. Palbo has been tested in other disease including dedifferentiated and well-differentiated liposarcoma where CDK4 is amplified. In this disease Palbo has been reported to increase the PFS at 12 weeks to 66% surpassing the primary end of 40% in patients who had progressed on at least one primary regimen. The major toxicity is neutropenia. Other CDK4 inhibitors are in clinical trial, including LEE001 (Novartis) and LY2835219 (Eli Lilly). These agents exhibit a somewhat different toxicity profile with neutropenia and QTc prolongation with LEE001 and gastrointestinal toxicity with LY2835219. Recently, CDK4 inhibition has been shown to induce either a reversible cell cycle arrest or an irreversible senescent state. The proteolytic turnover of MDM2 appears to be critical for cellular senescence induced by CDK4 inhibition in liposarcoma, breast, lung and glioma cell lines. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. In a small series of liposarcoma patients treated with Palbo the suppression of MDM2 expression correlated with improved clinical outcome suggesting that loss of MDM2 by inhibition of CDK4 could be a predictive biomarker for improved clinical outcome with this class of drugs. Citation Format: Gary K. Schwartz. The Targeting of CDK4/6: Have we gone full circle? [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA04.

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.

G protein pathway suppressor 2 (GPS2) acts as a tumor suppressor in liposarcoma.

Liposarcoma(LPS) is the most common type of soft tissue sarcoma accounting for 20 % of all adult sarcomas. However, the molecular pathogenesis of this malignancy is still poorly understood. Here, we showed that GPS2 expression was downregulated in LPS and correlated with the prognosis of this disease. In vitro study showed that knockdown of GPS2 resulted in enhanced proliferation and migration of LPS cell line SW872, without significant influence of cell death. Conclusively, our results suggest that GPS2 may act as a tumor suppressor in LPS and serve as a potential prognosis marker for this disease.

Abstract 1284: Lurbinectedin reduces tumor-associated macrophages and the production of inflammatory cytokines, chemokines, and angiogenic factors in preclinical models

Abstract Lurbinectedin, currently undergoing clinical evaluation in ovarian, breast and small-cell lung cancer patients, inhibits active transcription. The drug is structurally related to trabectedin containing the same pentacyclic skeleton of the fused tetrahydroisoquinoline rings, but differing by the presence of a tetrahydro-B-carboline replacing the additional tetrahydroisoquinoline of trabectedin. We investigated whether lurbinectedin has the ability to regulate the inflammatory tumor microenvironment in vitro and in vivo. Human purified monocytes were highly sensitive to lurbinectedin (IC50: 5-10 nM) and, after drug treatment, underwent caspase-8-dependent apoptosis. Furthermore, in vitro, lurbinectedin significantly inhibited the production of selected inflammatory chemokines (CCL2, CXCL8) and VEGF by stimulated monocytes and liposarcoma cell lines. Administration of lurbinectedin to mice bearing a murine fibrosarcoma -resistant to this compound in vitro- resulted in significant anti-tumor activity (T/C value around 50%). The analysis of immune cells of blood spleen and tumor tissues during treatment with lurbinectedin revealed a significant and selective decrease in the subset of monocytes and macrophages, including tumor-associated macrophages (TAM). A gene expression analysis of monocytes treated with lurbinectedin indicated a modulation of the transcriptional program in LPS-stimulated human monocytes. Overall, these results indicate that lurbinectedin affects the inflammatory micro-environment, with a selective apoptotic-inducing effect on mononuclear phagocytes and a specific inhibition of production of inflammatory cytokines. Citation Format: Paola Allavena, Cristina Belgiovine, Manuela Liguori, Ezia Bello, Roberta Frapolli, Carlos M. Galmarini, Maurizio D’Incalci. Lurbinectedin reduces tumor-associated macrophages and the production of inflammatory cytokines, chemokines, and angiogenic factors in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1284.

Abstract 1634: The RALA pathway drives invasion and metastasis of soft tissue sarcomas by regulating MMP14 localization and activity

Abstract Soft tissue sarcomas (STS) are a heterogeneous group of tumors originating from mesodermal tissues such as skeletal muscle, adipose, and fibrous tissue. The propensity of STS to metastasize, particularly to the lung which occurs in approximately 20% of all cases, is the major source of mortality associated with this disease. Currently, themolecular mechanisms that drive STS metastasis are poorly understood greatly limiting our ability to determine which STS will metastasize and impeding the development of targeted therapies for the treatment of STS metastases. In this study we utilized analysis of publicly available gene expression data to identify loss of PPP2R1B expression as a strong prognosticator of STS metastasis. In vitro analyses using SW872 liposarcoma (LPS) and HT1080 fibrosarcoma cell lines determined that PPP2R1B plays an essential role in the dephosphorylation and inactivation of RALA, a major downstream effector of the Ras pathway. RALA was found to be essential for invasion of STS cells in modified Boyden chamber assays as knockdown of RALA by either siRNA or shRNA significantly decreased invasion through matrigel. Analysis of publicly available gene expression data revealed that RALA expression correlated with more aggressive LPS subtypes and was prognostic of LPS metastasis. Furthermore, we identify the transmembrane matrix metalloprotease MMP14 (MT1-MMP) as a key downstream effector of RALA mediated invasion in STS. Stable knockdown of RALA in SW872 and HT1080 cells resulted in significant mislocalization of MMP14 and reduced MMP14 enzymatic activity. MMP14 expression was also significantly prognostic of metastasis in a publically available LPS cohort. Finally, we demonstrate that a three gene signature comprised of PPP2R1B, RALA, and MMP14 can retrospectively stratify LPS patients into groups of low, moderate, or high risk for metastatic recurrence. To the best of our knowledge this is the first demonstration of a vital role for PPP2R1A and RALA in STS invasion and metastasis and these data demonstrate a novel role for RALA in the localization and activity of MMP14. In conclusion, we have identified the PPP2R1B-RALA-MMP14 axis as driver of STS metastasis with potential clinical value as both a prognostic marker and therapeutic target. Citation Format: Steven T. Sizemore, Fen Xia, Arnab Chakravarti. The RALA pathway drives invasion and metastasis of soft tissue sarcomas by regulating MMP14 localization and activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1634.

Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma

Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation.

Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3.

The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Potential targeted therapeutic approaches in liposarcoma

Liposarcoma (LPS) is rare malignant tumor of fat cells in deep soft tissue that affects adults between the ages of 40 and 60 [1]. This tumor is extremely aggressive with high morbidity and mortality. World Health organization classifies LPS into five subtypes: well-differentiated (WDLPS)/Atypical lipomatous tumors (ALT); dedifferentiated (DDLPS); myxoid (MLPS); pleomorphic (PLPS); and mixed type LPS [2]. Most common modality of treatment is surgical removal of tumor followed by radiation and chemotherapy. The five year survival rate of LPS patients vary from 56%-100% based on different subtypes [3]. Previous genetic studies have focused either on a subset of target genes or one subtype of LPS. Till now no reports have provided a detailed genomic analysis of LPS which included all different subtypes of LPS. Therefore, recently we defined the genomic landscape of LPS using SNP array and whole exome sequencing and identified a spectrum of altered genes and pathways in different subtypes of LPS patients and cell lines [4]. WDLPS and DDLPS subtypes have a characteristic feature of amplified region of chromosome 12q13-15 containing several well-known oncogenes such as MDM2, CDK4 and HMGA2 [5]. SNP array analysis of LPS patients and cell lines identified recurrent amplification of Carboxypeptidase M (CPM) gene only in WDLPS and DDLPS subtypes. We studied in detail CPM gene in vitro and in vivo and showed its involvement in liposarcomagenesis. CPM is a membrane bound enzyme and we found it expressed on the cell surface of LPS cells suggesting it as a potential therapeutic target. Carboxypeptidase activity of CPM is known to regulate the processing of EGF [6]. We found downregulation of CPM expression leads to reduced EGFR signalling and induces apoptosis of LPS cells. Future studies are ongoing detailing further role of enzymatic activity of CPM in EGFR signaling and enhancement of tumor growth. Further, we are developing both a quantitative PCR assay and an ELISA to measure CPM activity in the serum to develop a biomarker to measure minimal residual disease which might aid in monitoring therapy. Specific and selective inhibitors of CPM are not available in the market; therefore, we are also aiming to do screening to find selective and specific small molecules to inhibit CPM enzymatic activity. We proposed CPM as potential therapeutic candidate which could be used as targeted approach to manage CPM amplified WDLPS or DDLPS patients. Next, whole exome sequencing and targeted exome sequencing identified various known cancer related and novel genes recurrently mutated in different subtypes of LPS. MAPK, ErbB, JAK-STAT, Wnt, apoptosis, cell cycle, DNA replication and repair and axon guidance pathways were found to be potentially involved in liposarcomagenesis. We identified recurrent mutations in previously unidentified genes in LPS associated with DNA damage repair pathways. LPS tumors with DNA repair mutations could be completely dependent on other backup repair pathways for the survival which may be exploited to induce synthetic lethality as therapeutic approach in these tumors. Interestingly, we reported for the first time multi-region genomic analysis of single LPS patient's tumor signifying intra-tumor mutational and copy number heterogeneity. The current basis for most of the personalized medicine approaches depends on the genomic landscape of a single tumor biopsy sample. Due to the frequent large size of LPS tumors compared to other solid tumors, intra-tumor heterogeneity will lead to difficulties in identifying biomarkers and therapeutic targets. Our preliminary analysis of intra-tumor heterogeneity mandates the need for future detailed studies exploring the evolution of LPS tumors leading to progression. In summary, our recent work gave insights into global genomic spectrum of LPS cohort for development of novel therapeutic strategies and for understanding the pathogenesis this deadly disease.

Tumor-associated fibroblasts promote the proliferation and decrease the doxorubicin sensitivity of liposarcoma cells.

The reasons for the distinct chemoresistance of liposarcomas and their high risk of local recurrence still remain unclear. Depending on the histological subtype of liposarcoma, first-line therapy with the cytostatic agent, doxorubicin, only achieves response rates of approximately 36%. Approximatley 70% of all local recurrences develop in spite of complete surgical resection of the primary tumor with microscopically negative margins. In this study, we aimed to assess the influence of tumor-associated fibroblasts (TAFs) obtained from surgically removed liposarcomas on the well-established human liposarcoma SW872 cell line. Intratumoral TAFs were isolated from intermediate- and high-grade liposarcoma samples. The human liposarcoma cell line, SW872, was co-cultured with the corresponding TAFs or with dermal fibroblasts as a control. The proliferation (by BrdU assay), cell viability (by MTT assay) and sensitivity to doxorubicin (using the iCELLigence system) of the co-cultured SW872 cells were examined. The SW872 cells exhibited a significant increase in proliferation and viability when co-cultured with the TAFs. As detected by real-time cell analysis, the SW872 cells co-cultured with the TAFs exhibited a diminished response towards doxorubicin. Notably, co-culture with TAFs obtained from high-grade liposarcoma samples resulted in higher proliferation and increased chemoresistance than co-culture with TAFs obtained from intermediate-grade liposarcoma samples. The findings of the present study thus indicate that TAFs from liposarcomas enhance the proliferation and decrease the chemosensitivity of SW872 liposarcoma cells significantly compared with normal fibroblasts from the dermis. TAFs from more malignant liposarcomas promoted tumor cell proliferation and chemoresistance more strikingly than TAFs from less malignant liposarcomas. These data provide evidence for the influence of the tumor microenvironment on liposarcoma and support for further investigations in patients with different liposarcoma subentities, assessing the influence of TAFs on tumor progression.

Preclinical activity of selinexor, an inhibitor of XPO1, in sarcoma.

Selinexor is an orally bioavailable selective inhibitor of nuclear export that has been demonstrated to have preclinical activity in various cancer types and that is currently in Phase I and II clinical trials for advanced cancers. In this study, we evaluated the effects of selinexor in several preclinical models of various sarcoma subtypes. The efficacy of selinexor was investigated in vitro and in vivo using 17 cell lines and 9 sarcoma xenograft models including gastrointestinal stromal tumor (GIST), liposarcoma (LPS), leiomyosarcoma, rhabdomyosarcoma, undifferentiated sarcomas, and alveolar soft part sarcoma (ASPS). Most sarcoma cell lines were sensitive to selinexor with IC50s ranging from 28.8 nM to 218.2 nM (median: 66.1 nM). Selinexor suppressed sarcoma tumor xenograft growth, including models of ASPS that were resistant in vitro. In GIST cells with KIT mutations, selinexor induced G1- arrest without attenuation of phosphorylation of KIT, AKT, or MAPK, in contrast to imatinib. In LPS cell lines with MDM2 and CDK4 amplification, selinexor induced G1-arrest and apoptosis irrespective of p53 expression or mutation and irrespective of RB expression. Selinexor increased p53 and p21 expression at the protein but not RNA level, indicating a post-transcriptional effect. These results indicate that selinexor has potent in vitro and in vivo activity against a wide variety of sarcoma models by inducing G1-arrest independent of known molecular mechanisms in GIST and LPS. These studies further justify the exploration of selinexor in clinical trials targeting various sarcoma subtypes.