Human Leiomyosarcoma Research Articles

Immune evasion in lung metastasis of leiomyosarcoma: upregulation of EPCAM inhibits CD8+ T cell infiltration

BackgroundLeiomyosarcomas are among the most common histological types of soft tissue sarcoma (STS), with no effective treatment available for advanced patients. Lung metastasis, the most common site of distant metastasis, is the primary prognostic factor. We analysed the immune environment targeting lung metastasis of STS to explore new targets for immunotherapy.MethodsWe analysed the immune environment of primary and lung metastases in 38 patients with STS using immunohistochemistry. Next, we performed gene expression analyses on primary and lung metastatic tissues from six patients with leiomyosarcoma. Using human leiomyosarcoma cell lines, the effects of the identified genes on immune cells were assessed in vitro.ResultsImmunohistochemistry showed a significant decrease in CD8+ cells in the lung metastases of leiomyosarcoma. Among the genes upregulated in lung metastases, epithelial cellular adhesion molecule (EPCAM) showed the strongest negative correlation with the number of CD8+ cells. Transwell assay results showed that the migration of CD8+ T cells was significantly increased in the conditioned media obtained after inhibition or knock down of EPCAM.ConclusionsEPCAM was upregulated in lung metastases of leiomyosarcoma, suggesting inhibition of CD8+ T cell migration. Our findings suggest that EPCAM could serve as a potential novel therapeutic target for leiomyosarcoma.

MiR-130b modulates the invasive, migratory, and metastatic behavior of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive, often poorly differentiated cancer of the smooth muscle (SM) lineage for which the molecular drivers of transformation and progression are poorly understood. In microRNA (miRNA) profiling studies, miR-130b was previously found to be upregulated in LMS vs. normal SM, and down-regulated during the differentiation of mesenchymal stem cells (MSCs) into SM, suggesting a role in LMS tumor progression. In the present study, the effects of miR-130b on human LMS tumorigenesis were investigated. Stable miR-130b overexpression enhanced invasion of LMS cells in vitro, and led to the formation of undifferentiated, pleomorphic tumors in vivo, with increased growth and metastatic potential compared to control LMS cells. TSC1 was identified as a direct miR-130b target in luciferase-3'UTR assays, and shRNA-mediated knockdown of TSC1 replicated miR-130b effects. Loss-of-function and gain-of-function studies showed that miR-130b levels regulate cell morphology and motility. Following miR-130b suppression, LMS cells adopted a rounded morphology, amoeboid mode of cell movement and enhanced invasive capacity that was Rho/ROCK dependent. Conversely, miR-130b-overexpressing LMS cells exhibited Rho-independent invasion, accompanied by down-regulation of Rho-pathway effectors. In mesenchymal stem cells, both miR-130b overexpression and TSC1 silencing independently impaired SM differentiation in vitro. Together, the data reveal miR-130b as a pro-oncogenic miRNA in LMS and support a miR-130b-TSC1 regulatory network that enhances tumor progression via inhibition of SM differentiation.

Abstract B005: Hypoxic sarcoma spheroid on a chip: Insights into treatment response

Abstract Context: Hypoxia is found in at least 47% of soft-tissue sarcoma (STS) patients and is known to be a major contributor to treatment resistance, metastasis progression and associated with poor prognosis. Indeed, expression of hypoxic proteins (HIF1-α, CAIX) in STS is associated with a shorter overall survival, and a shorter progression-free survival in patients. Specifically, Yang et al. demonstrated in 2017 that patients stratified by a 24-gene hypoxia signature had a poorer distant metastasis free survival. Nevertheless, hypoxia is seldom considered during treatment development due to the lack of a user-friendly way to culture naturally hypoxic 3D tumor models. Therefore, we developed a user-friendly in vitro preclinical tool allowing the study of natural chronic hypoxia and its effect on treatment response. Methods: Microfabrication technologies were used to design a microfluidic chip allowing culture, maintenance, treatment, and analysis of 240 STS naturally hypoxic spheroids (750µm of diameter). SK-LMS-1 Human leiomyosarcoma and STS117 (human primary undifferentiated pleomorphic sarcoma cells) spheroids were formed 48h after seeding. Gold-standard hypoxic protein Hypoxia Inducible Factor 1 alpha (HIF1- α) and Carbonic Anhydrase IX (CAIX) expression was assessed by Western Blot (WB). Spatial distribution of CAIX was assessed by immunofluorescence (IF). Based on the IF, an in silico model of the oxygen consumption of the spheroids was built using COMSOL. As a proof of concept, the spheroids were treated using two oxygen-dependent treatment: RT and hypoxia prodrug Tirapazamine (TPZ). DNA-damages were quantified using yH2AX in IF to assess treatment efficacy. Results: These spheroids on-a-chip are the largest to date (>750µm), and express gold-standard hypoxic protein CAIX in their core only, a feature absent in spheroids of 450µm of diameter of the same cell lines. HIF1-α expression patterns were cell line dependent. CAIX expression in SK-LMS-1 and STS117 large spheroids was respectively 7.8 and 24 times higher than in 450µm spheroids. Quantification of DNA damages (count of yH2AX foci/nuclei area in mm²) demonstrated that TPZ preferentially targets the hypoxic core. However, the response (DNA damages) to RT alone showed no evidence of radioresistance of the hypoxic regions, on samples fixed 24h after irradiation. A possible explanation could be that, in 24h, the oxygen-dependent DNA repair and the accumulation of DNA damages in hypoxic regions hide the oxygen-dependent radioresistance. Therefore, quantification of DNA damages with yH2AX 30min after irradiation is currently being investigated, as maximum DNA damage response has been shown to be observed at this time point. Our 750µm spheroids naturally display a hypoxic core expressing gold-standard hypoxic protein CAIX at 120µm of depth, a feature absent in smaller spheroids. Together, these results cement our microfluidic device and our spheroids as a potent fundamental and pre-clinical tool to explore the biology of hypoxia on 3D tumor model, its effects on treatment response. Citation Format: Elena Refet-Mollof, Ouafa Najyb, Rodin Chermat, Audrey Glory, Julie Lafontaine, Philip Wong, Thomas Gervais. Hypoxic sarcoma spheroid on a chip: Insights into treatment response [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B005.

Case Report: Giant Multiloculated Pseudocystic Jejunal Leiomyosarcoma in a Dog: Atypical Morphologic Features of Canine Intestinal Leiomyosarcoma

A 10-year-old intact female Rottweiler dog weighing 29 kg presented with 2 days history of vomiting, anorexia, and lethargy to KonKuk University Teaching Hospital, Seoul, South Korea. Ultrasonography and computed tomography (CT) scannings revealed a well-demarcated, large mass (29 × 19 × 11 cm) with numerous fluid-filled cavities. Metastases to adjacent lymph nodes were also identified on CT. This large mass and the affected intestinal segments were excised for palliative purposes. Postoperatively, the dog recovered uneventfully without any complications. The cut surface of the mass showed an exophytic growth pattern of multiloculated cystic lesions filled with serosanguineous fluid, large cavities filled with necrotic exudate, and fistulous connections between the intestinal lumen and the necrotic cavity in the mass. On histopathology, the mass was a spindle cell neoplasm expanding from the jejunal muscular layer and with pseudocystic changes. Additional immunohistochemical analysis using antibodies against smooth muscle actin, desmin, and CD-117 demonstrated that the mass was consistent with a leiomyosarcoma. Six months post-operatively, plain radiography revealed an abdominal mass, suspected to be recurrence from jejunal leiomyosarcoma. The owner decided to euthanize the dog due to financial constraints. This case report describes the atypical morphology and clinical progression of a large canine jejunal leiomyosarcoma, which had similar clinical features as those of human leiomyoma and leiomyosarcoma.

A nutrient mixture reduced tumor growth of SK-UT-1 human leiomyosarcoma cells in vivo and in vitro by inhibiting MMPs and inducing apoptosis.

Uterine leiomyosarcoma is a rare malignant smooth muscle tumor originating in the uterine wall that generally responds poorly to chemotherapy and radiation. We investigated the in vitro effects of a novel nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract on the human leiomyosarcoma cell line SK-UT-1by measuring cell proliferation, invasiveness, apoptosis, and expression of matrix metalloproteinases (MMP). We also tested the effects of nutrient mixture in vivo using nude mice. Human leiomyosarcoma SK-UT-1cells were treated with different concentrations of nutrient mixture. Cell proliferation was determined by MTT assay; MMP expression by gelatinase zymography; invasion by Matrigel assay; migration by scratch test; apoptosis using Live Green caspase kit. In vivo studies were conducted on 5-6weeks old female nude mice inoculated subcutaneously with 3• 106SK-UT-1cells. The mice were fed a regular diet or a diet supplemented with 0.5% nutrient mixture. After four weeks, the mice were sacrificed and the tumors were weighed and processed for histology. In vitro, nutrient mixture treatment was not toxic to SK-UT-1cells at 250µg/ml but exhibited 20% and 40% cytotoxicity at 500and 1000µg/ml respectively. Zymography did not show bands for either MMP-2or MMP-9in SK-UT-1cells. However, treatment with phorbol myristate acetate stimulated the expression of MMP-9, both active and inactive forms in equal proportion. Nutrient mixture inhibited the secretion of both active and inactive forms in a dose dependent manner. Invasion through Matrigel and migration by scratch test were inhibited in a dose dependent fashion, with both invasion and migration inhibited at 250µg/ml. Live Green Caspase apoptosis assay demonstrated slight apoptosis at 100µg/ml and significant apoptosis at 250to 1000µg/ml. The results of in vitro studies were further confirmed in vivo by showing 50% decrease in tumor weight, 40% reduction in tumor burden compared to the tumors from mice fed regular diet. The results suggest a therapeutic potential for nutrient mixture in uterine leiomyosarcoma treatment.

Molecular Bio-Imaging Probe for Non-Invasive Differentiation Between Human Leiomyoma Versus Leiomyosarcoma.

Leiomyosarcoma is the most frequent subtype of the deadly uterine sarcoma and shares many common clinical grounds with leiomyoma, which is in turn the most common solid benign uterine neoplasm. With the recent progress in minimally invasive techniques for managing leiomyomas, accurate preoperative diagnosis of uterine masses has become the most important selection criterion for the safest therapeutic option. Therefore, different imaging modalities would be playing a key role in management of uterine masses. Testing for a sarcoma-specific promoter that expresses its downstream reporter gene only in leiomyosarcoma and not in leiomyoma or healthy uterine tissue. Adenoviral vectors were utilized both in vitro and in vivo to test the specificity of the promoters. Quantitative studies of downstream gene expression of these promoters was carried out both in vitro and in vivo. Our data indicated that human leiomyosarcoma cells highly expressed the reporter gene downstream to survivin promoter (Ad-SUR-LUC) when compared with benign leiomyoma or normal cells (p value of 0.05). Our study suggested that survivin is the unique promoter capable of distinguishing between the deadly sarcoma and the benign counterparts.

1730P - Influence of eribulin on proliferation, migration and invasion properties of leiomyosarcoma cell line models

BackgroundEribulin has an effect on proliferation, migration and invasion of sarcoma and breast cancer in vitro assays. A phase III clinical trial in patients with advanced liposarcoma (LPS) or leiomyosarcoma (LMS) showed an improvement in overall survival (OS) for the eribulin arm compared to dacarbazine, although no benefit was observed in the LMS restricted subgroup. Based on these results, eribulin was approved only for advanced LPS patients. The aim of the current study is to evaluate the effect of eribulin on key cell properties, like proliferation, migration, and invasion in LMS cell lines, in conventional 2D, but also 3D culture models. MethodsEribulin was kindly provided by Eisai Inc. (Andover, USA) and tested on CP0024 and SK-UT-1 human LMS cell lines. Cellular proliferation was determined by SRB assay on 2D, and measured by spheroid diameter and by propidium iodide and calcein-AM double staining in 3D cultures by Celigo Platform (Nexcelom, Lawrence, USA).Cells were treated with increased concentrations of eribulin to determine IC50 values and its effect on proliferation. Chemotaxis and invasion were determined using transwell assay on 2D culture. Spheroids grown during 4 days were laid or embedded in matrigel for 3D assays and quantified over 3-7 additional days. ResultsBoth cell lines had a mesenchymal-like appearance on monolayer culture, although SK-UT-1 had a more compact spheroid pattern compared to CP0024. Proliferation, migration, and invasion were examined after exposure to eribulin. The results showed that eribulin is active on both cell lines inhibiting proliferation in 2 and 3D culture conditions at concentrations below the nanomolar range. Moreover, eribulin significantly inhibited migration and invasion. To our knowledge, these findings are the first to demonstrate the effect of eribulin in LMS cell line 3D models. ConclusionsOur results support the in vitro activity of eribulin in LMS cell lines and its further exploration as an agent with clinical benefit in patients with LMS. Legal entity responsible for the studyThe authors. FundingEisai. DisclosureM. Mendiola: Research grant / Funding (institution), Travel / Accommodation / Expenses: Eisai. A. Gallego Martínez: Travel / Accommodation / Expenses, Non-remunerated activity/ies, training courses: Eisai. A. Redondo: Honoraria (self), Research grant / Funding (institution): Eisai. All other authors have declared no conflicts of interest.

1659P - Combination of eribulin plus gemcitabine in L-sarcomas

Background: Eribulin (Halaven®; Eisai Co., Tokyo, Japan) is a synthetic analogue of halichondrin B that was approved for advanced liposarcoma. Eribulin, besides its well stablished cytotoxic activity, induces vascular remodelling and increased tumor perfusion, as well as inhibits pericyte- and endothelial-driven angiogenesis, which could facilitate the distribution of other drugs within the tumor. Altogether, we hypothesize that eribulin and gemcitabine, acting in different cell cycle phases and modulating distintly tumour microenvironment, could be synergistic, without overlapping toxicity. Methods: CP0024 human leiomyosarcoma primary cell line, SK-UT-1 human leiomyosarcoma cell line and, 93T449 and 94T778 human liposarcoma cell lines were treated with increased concentrations of eribulin (1x10-7M to 1x10-11M) or gemcitabine (1x10-9M to 1x10-13M) to determine IC50 values. Combination index values of eribulin plus gemcitabine were calculated, after treating L-sarcoma cell lines with both drugs using the following administration sequences: concomitant (E+G), eribulin followed by gemcitabine (E→G) and gemcitabine followed by eribulin (G→E). Cell viability, at 72 hours, was measured by MTS cell proliferation assay. Results: The IC50 values determined for eribulin ranged between 0.28nM (SK-UT-1) and 1.98nM (94T778), while IC50 values of gemcitabine ranged between 2.10nM (SK-UT-1) and 7.02nM (94T778). The combination index values at ED50 (the effective doses at which 50% of cell killing occurred) ranged: between 0.27 (SK-UT-1) and 0.89 (94T778), when cell lines were treated with E+G; between <0.10 (SK-UT-1) and 0.33 (CP0024) when treated with G→E; and between <0.10 (SK-UT-1) and 0.20 (CP0024) when cells were treated with E→G. All the cell lines showed a higher synergism when treated using the E→G administration sequence. Conclusions: Eribulin and gemcitabine combination is synergic in L-sarcoma cell lines. The administration of eribulin, followed by gemcitabine is the recommended administration sequence for further studies. The mechanisms underlying eribulin plus gemcitabine synergy, as well as predictive biomarkers of response to the combination, will be evaluated in in vitro and in vivo models of L-sarcoma. Legal entity responsible for the study: Instituto de Biomedicina de Sevilla. Funding: Eisai Co., Ltd. Disclosure: D.S. Moura: Corporate-sponsored research fees: Eisai. Co. Ltd. J. Martin-Broto: Corporate-sponsored research fees: Eisai. Co., Ltd., Novartis, PharmaMar; Honoraria or consultation fees: Lilly, Novartis, PharmaMar; Company sponsored speaker’s bureau: PharmaMar. All other authors have declared no conflicts of interest.

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates.

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.

Effect of 2-methoxyestradiol on SK-LMS-1 uterine leiomyosarcoma cells.

An endogenous metabolite of 17β-estradiol, 2-methoxyestradiol (2-ME), has affinity for estrogen receptors. This compound was reported to be a promising antitumor drug due to its anti-proliferative effects on a wide range of tumor cell types. Numerous previous studies have been performed to evaluate the cytotoxic effects of 2-ME on tumor cell lines in following the induction of G2/M cell cycle arrest and subsequent apoptosis. Uterine leiomyosarcoma (ULMS) is a relatively rare malignant smooth muscle cell tumor that develops in the uterus muscle layer. The aim of the present study was to examine the in vitro anti-proliferative effects of 2-ME on SK-LMS-1 human leiomyosarcoma cells. An MTT assay, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay, immunocytochemistry and western blotting were performed. A high concentration (10−5 M) of 2-ME was identified to have an anti-proliferative effect on SK-LMS-1 cells. Additionally, expression of the apoptosis markers was upregulated in the presence of 10−5 M 2-ME, according to western blot analysis. Furthermore, the expression level of an autophagic marker, light chain 3, was increased by 2-ME treatment in a dose-dependent manner. This was associated with cell death induced by the upregulation of phosphorylated extracellular-signal-regulated kinase 1/2 signaling pathway. The results of the present study demonstrated that 2-ME, which is used as a therapeutic agent for treating solid tumors, exhibits apoptotic and anti-proliferative effects depending on the dose. Therefore, 2-ME may be a potential therapeutic reagent for human ULMS, but the appropriate dose of this compound should be carefully selected.

Activation of the cell membrane angiotensin AT2 receptors in human leiomyosarcoma cells induces differentiation and apoptosis by a PPARγ - dependent mechanism.

Angiotensin II (Ang II), the main effector peptide of the renin-angiotensin system (RAS), acting on AT1 and AT2 receptors participates in the regulation of proliferation, differentiation and apoptosis in tumour cells. The peroxisome-proliferator activated receptor γ (PPARγ) and its ligands exert anti-tumour effects in various human cancer cell lines. The present study investigates the effects initiated by AT1- and AT2 receptor stimulation in SK-UT-1 cells, a human leiomyosarcoma cell line, and clarifies the role of the PPARγ in the AT2 receptor-induced differentiation and apoptosis.Selective stimulation of AT1- and AT2 receptors was achieved by incubation of the cells with Ang II (10-6 M) in the presence of the selective AT2 receptor antagonist, PD 123177 (10-6 M) and the AT1 receptor antagonist, losartan (10-5 M), respectively, the selective PPARγ antagonist, GW 9662, was used at concentration 10-6 M. The expression of smooth muscle cell differentiation markers, SM22α and calponin, was analysed at RNA- and protein levels using RT PCR and Western blot, which was also used to quantify Bcl-2-, Bax- and cleaved caspase-3 proteins. The translocation of the AT2-receptor interacting protein 1 (ATIP1) to the nuclei was studied by Western blot and immunofluorescence staining. The mitochondrial status and the metabolic activity in response to AT1- and AT2 receptor activation were assessed by the quantification of 99mTc - sestamibi and 2´-deoxy-2´-[18F]fluoro-D-glucose uptake.AT1 receptor stimulation did not exert any profound effects in quiescent SK-UT-1 cells. The effects induced by Ang II acting on AT2 receptors were time-dependent. A short, 3 - 6 h lasting stimulation promotes differentiation, i.e increases in the mRNA- and protein levels of SM22α and calponin, whereas a sustained stimulation for 48 h activates the intrinsic apoptotic pathway, as evidenced by reduced cell numbers, down-regulation of the anti-apoptotic Bcl-2 protein and increased levels of the Bax protein and cleaved caspase-3. The effects were reversed by the PPARγ antagonist, GW 9662, clearly implying a PPARγ-dependent mechanism. Our results also demonstrate a co-localisation of the AT2-receptor interacting protein, ATIP1, and the PPARγ in nuclei of SK-UT-1 cells and an accumulation of ATIP1 in the nuclear fraction in response to AT2 receptor stimulation. The regulation of the differentiation and apoptosis via the AT2 receptor favours an important functional role of this receptor in quiescent, slow-cycling SK-UT-1 cells and provides the rationale for the use of AT1 receptor antagonists for the treatment of human leiomyosarcomas.

Dual inhibition of the PI3K/AKT/mTOR pathway suppresses the growth of leiomyosarcomas but leads to ERK activation through mTORC2: biological and clinical implications.

The PI3K/AKT/mTOR pathway plays a crucial role in the development of leiomyosarcomas (LMSs). In this study, we tested the efficacy of dual PI3K/mTOR (BEZ235), PI3K (BKM120) and mTOR (everolimus) inhibitors in three human LMS cell lines. In vitro and in vivo studies using LMS cell lines showed that BEZ235 has a significantly higher anti-tumor effect than either BKM120 or everolimus, resulting in a greater reduction in tumor growth and more pronounced inhibitory effects on mitotic activity and PI3K/AKT/mTOR signaling. Strikingly, BEZ235 but neither BKM120 nor everolimus markedly enhanced the ERK pathway. This effect was reproduced by the combination of BKM120 and everolimus, suggesting the involvement of mTORC2 via a PI3K-independent mechanism. Silencing of RICTOR in LMS cells confirmed the role of mTORC2 in the regulation of ERK activity. Combined treatment with BEZ235 and GSK1120212, a potent MEK inhibitor, resulted in synergistic growth inhibition and apoptosis induction in vitro and in vivo. These findings document for the first time that dual PI3K/mTOR inhibition in leiomyosarcomas suppress a negative feedback loop mediated by mTORC2, leading to enhanced ERK pathway activity. Thus, combining a dual PI3K/mTOR inhibitor with MEK inhibitors may be a relevant approach to increase anti-tumor activity and prevent drug resistance in patients with LMS.

Melatonin suppression of aerobic glycolysis (Warburg effect), survival signalling and metastasis in human leiomyosarcoma.

Leiomyosarcoma (LMS) represents a highly malignant, rare soft tissue sarcoma with high rates of morbidity and mortality. Previously, we demonstrated that tissue-isolated human LMS xenografts perfused in situ are highly sensitive to the direct anticancer effects of physiological nocturnal blood levels of melatonin which inhibited tumour cell proliferative activity, linoleic acid (LA) uptake and metabolism to 13-hydroxyoctadecadienoic acid (13-HODE). Here, we show the effects of low pharmacological blood concentrations of melatonin following oral ingestion of a melatonin supplement by healthy adult human female subjects on tumour proliferative activity, aerobic glycolysis (Warburg effect) and LA metabolic signalling in tissue-isolated LMS xenografts perfused in situ with this blood. Melatonin markedly suppressed aerobic glycolysis and induced a complete inhibition of tumour LA uptake, 13-HODE release, as well as significant reductions in tumour cAMP levels, DNA content and [(3) H]-thymidine incorporation into DNA. Furthermore, melatonin completely suppressed the phospho-activation of ERK 1/2, AKT, GSK3β and NF-kB (p65). The addition of S20928, a nonselective melatonin antagonist, reversed these melatonin inhibitory effects. Moreover, in in vitro cell culture studies, physiological concentrations of melatonin repressed cell proliferation and cell invasion. These results demonstrate that nocturnal melatonin directly inhibited tumour growth and invasion of human LMS via suppression of the Warburg effect, LA uptake and other related signalling mechanisms. An understanding of these novel signalling pathway(s) and their association with aerobic glycolysis and LA metabolism in human LMS may lead to new circadian-based therapies for the prevention and treatment of LMS and potentially other mesenchymally derived solid tumours.

Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo

Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.

156 EFFECT OF 2-METHOXYESTRADIOL (2-ME) ON HUMAN UTERINE LEIOMYOSARCOMA

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-oestradiol (E2) and has affinity for oestrogen receptors. It was reported as a promising antitumour drug due to antiproliferative activity in many tumour cell types. It has also been used in several preclinical and clinical studies for treatment of solid tumours. Thus, several studies have been conducted to investigate the cytotoxic effect of 2-ME on tumour cell lines in which it induced G2/M cell cycle arrest and subsequent apoptosis. Uterine leiomyosarcoma is a rare form of malignant smooth muscle cell tumour in the uterus muscle layer. This tumour is also influenced by oestrogen, which acts as a promoter. The purpose of this study was to examine the antiproliferative effect of 2-ME in vitro and in vivo. To perform in vitro experiments, we evaluated the antiproliferative effect of 2-ME on SK-LMS-1 cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and Western blot. In vivo experiment also investigated the effect of 2-ME on uterine leiomyoma by measuring tumour size and Western blotting. Employing the mouse xenograft model using human leiomyosarcoma SK-LMS-1 cell line compared with the positive control flavopiridol for 2 weeks, mice were divided into 4 groups (6 mice per each group); VE, 2-ME (75 or 150 mg kg–1), or flavopiridol (7.5 mg kg–1). All experimental procedures were performed in triplicate. P-values were calculated using one-way ANOVA, followed by Tukey’s test for multiple comparisons of columns. Data were considered statistically significant at P &lt; 0.05. In vitro, we confirmed that 2-ME at 10–5 M and flavopiridol have an antiproliferative influence on SK-LMS-1 cells, but the same effect at 10–7 M or 10–6 M dose of 2-ME were not detected. These 2 doses showed little proliferative response in the MTT assay. Also, BAX/BCL-2 and LC3 expression were increased by 2-ME (10–5 M) treatment in Western blot analysis. In the in vivo xenograft study, application of 2-ME (75 mg kg–1) resulted in an increased tumour size but 150 mg kg–1 of 2-ME and flavopiridol decreased the size. In a previous study, we found that 2-ME has 2 types of action in an in vitro and an in vivo model. The 2-ME, which is used as a therapeutic agent for solid cancers, has not only apoptotic but also a proliferative effect, depending on the dose. We expect that 2-ME may be a potential therapeutic reagent for human uterine leiomyoma, but the appropriate dose of 2-ME should be used for oestrogen-response tumour treatment.

Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas.

BackgroundUSP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease.MethodsHeterozygous USP18 +/− FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined.ResultsUSP18 −/− FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6–12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441).ConclusionsUSP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1883-8) contains supplementary material, which is available to authorized users.

A Role for Versican in the Development of Leiomyosarcoma

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.

The inhibition of tyrosine kinase receptor signalling in leiomyosarcoma cells using the small molecule kinase inhibitor PTK787/ZK222584(Vatalanib®).

Leiomyosarcomas remain challenging tumors to manage and novel therapy strategies besides radiation and conventional chemotherapy are needed. Targeting angiogenesis by inhibition of vascular endothelial growth factor (VEGF) receptor tyrosine kinases (RTKs) of the tumor vasculature with small molecules is a promising new therapy. It has been shown recently that these receptors are not only expressed on tumor endothelium but also on tumor cells themselves. Thus, we investigated the expression of members of the VEGF receptor (VEGFR) family and corresponding growth factors in leiomyosarcoma tissue specimens and in the leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1. We evaluated the influence of the VEGFR inhibitor PTK787/ZK222584 (PTK787) on cell growth, migration, apoptosis and phosphorylation of intracellular signalling molecules. In human leiomyosarcoma tissue specimens VEGFR-1/-2 and platelet-derived growth factor receptor (PDGFR-β) were strongly expressed. Both leiomyosarcoma cell lines expressed VEGFR-1/-3 and PDGFR-β but VEGFR-2 protein expression was positive only in SK-UT-1. SK-LMS-1 and SK-UT-1 cells secreted high and low amounts of VEGF-A, respectively, whereas PDGF-BB secretion was similar in both cell lines. Application of PTK787 led to partial inhibition of PDGF-BB-activated AKT/p90RSK and ERK1/2 signalling pathways. In contrast, protein phosphorylation was not affected by PTK787 in VEGF-A-treated cells. PTK787 turned out to inhibit cell migration even though no effects were observed upon stimulation with VEGF-A or PDGF-BB. In line, cell growth in leiomyosarcoma cell lines remained unchanged upon PTK787 treatment alone and with subsequent VEGF-A- or PDGF-BB-stimulation. However, VEGF-A, but not PDGF-BB-treated cells showed increased cell death upon PTK787 treatment. VEGFR family members are expressed in leiomyosarcomas in vivo and in vitro. Upon receptor stimulation, PTK787 is able to inhibit subsequent phosphorylation events and influences cell survival but not metabolic activity and migration. Thus, the inhibitor is possibly an additional option in the treatment of leiomyosarcomas.

Reconstituted Human Myosin Light Chain Phosphatase Reveals Distinct Roles of Two Inhibitory Phosphorylation Sites of the Regulatory Subunit, MYPT1

The myosin light chain phosphatase (MLCP) is a cytoskeleton-associated protein phosphatase-1 (PP1) holoenzyme and a RhoA/ROCK effector, regulating cytoskeletal reorganization. ROCK-induced phosphorylation of the MLCP regulatory subunit (MYPT1) at two sites, Thr696 and Thr853, suppresses the activity, although little is known about the difference in the role. Here, we developed a new method for the preparation of the recombinant human MLCP complex and determined the molecular and cellular basis of inhibitory phosphorylation. The recombinant MLCP partially purified from mammalian cell lysates retained characteristics of the native enzyme, such that it was fully active without Mn2+ and sensitive to PP1 inhibitor compounds. Selective thio-phosphorylation of MYPT1 at Thr696 with ROCK inhibited the MLCP activity 30%, whereas the Thr853 thio-phosphorylation did not alter the phosphatase activity. Interference with the docking of phospho-Thr696 at the active site weakened the inhibition, suggesting selective autoinhibition induced by phospho-Thr696. Both Thr696 and Thr853 sites underwent autodephosphorylation. Compared with that of Thr853, phosphorylation of Thr696 was more stable, and it facilitated Thr853 phosphorylation. Endogenous MYPT1 at Thr696 was spontaneously phosphorylated in quiescent human leiomyosarcoma cells. Serum stimulation of the cells resulted in dissociation of MYPT1 from myosin and PP1C in parallel with an increase in the level of Thr853 phosphorylation. The C-terminal domain of human MYPT1(495–1030) was responsible for the binding to the N-terminal portion of myosin light meromyosin. The spontaneous phosphorylation at Thr696 may adjust the basal activity of cellular MLCP and affect the temporal phosphorylation at Thr853 that is synchronized with myosin targeting.

The tricyclic antidepressant amitriptyline is cytotoxic to HTB114 human leiomyosarcoma and induces p75NTR-dependent apoptosis

Nerve growth factor (NGF) receptors, TrKA and p75(NTR), are being investigated in cancer therapy. Our previous data show that, in HTB114 uterine leiomyosarcoma cells, p75(NTR)-dependent apoptosis is inducible by cytotoxic drugs and can suppress nerve growth factor-dependent growth. Although amitriptyline can kill cancer cells and bind TrKA/B, its effects on p75-dependent apoptosis are unknown. The aim of this paper was to evaluate the antineoplastic potential of amitriptyline, and the role of p75(NTR)-dependent apoptosis in the chemoresistant uterine HTB114 leiomyosarcoma. Using proliferation assays and fluorescence-activated cell sorting analysis, we found that amitriptyline caused a marked reduction in HTB114 cell viability, associated with the parallel upregulation of p75(NTR) expression. This converted the TrKA⁺-proliferating cells into TrKA⁺/p75(NTR⁺), leading to downregulation of TrKA-prosurvival signaling (AKT) and activation of p75(NTR)-dependent apoptosis (through caspase-3). Overall, we provide novel evidence that HTB114 uterine leiomyosarcoma cells are highly sensitive to amitriptyline, supporting the role of p75(NTR)-dependent apoptosis as a novel cytotoxic mechanism of this drug and of p75(NTR) as an inducible stress receptor and a novel target in clinical oncology.