Leiomyosarcoma Cell Line Research Articles

Treatment with epigenetic agents profoundly inhibits tumor growth in leiomyosarcoma.

Leiomyosarcomas are rare mesenchymal neoplasms characterized by a smooth muscle differentiation pattern. Due to the extremely poor prognosis in patients, the development of novel chemotherapeutic regimens remains critically important. In this study, multiple leiomyosarcoma cell lines, SK-UT1, SK-LMS1, and MES-SA were treated with varying doses of the DNA Methyltransferase Inhibitors (DNMTi) 5-azacitidine (Aza), 5-aza-2-deoxycytidine (DAC), and guadecitabine (SGI-110). The effect of these epigenetic modulators was measured using both in-vitro and in-vivo models.Of the three epigenetic modulators, Guadecitabine was the most effective at decreasing cell survival in LMS cell lines. SK-UT1 was found to be the more sensitive to all three epigenetic modulators, while SK-LMS1 and MES-SA were more resistant. The contrast in sensitivity seen was also represented by the increase in apoptosis in Aza and guadecitabine. In parallel with Aza, guadecitabine was observed to also arrest the cell cycle.Treatment with guadecitabine led to a decrease in growth across the spectrum of sensitivity in LMS cell lines, both in a delayed in vitro and in vivo model; in parallel experiments, apoptotic pathways were activated in sensitive and less sensitive lines. Additional studies are required to explore potential therapeutic applications and mechanisms for leiomyosarcoma treatment.

Abstract A35: Treatment with demethylating drugs inhibits tumor growth in leiomyosarcoma cell lines and xenograft models

Abstract Introduction: Leiomyosarcomas are rare mesenchymal neoplasms characterized by a smooth-muscle differentiation pattern. Patients show poor disease-specific survival due to high recurrence rates and low response to chemotherapy and radiation. Development of novel chemotherapeutic regimens is the need of the hour. Epigenetic mechanisms such as hypermethylation of gene promotor regions lead to abnormal gene silencing and have been found to influence the initiation and progression of cancer. Epigenetic drugs have been approved for treatment of various malignancies such as myelodysplastic syndrome, acute myeloid leukemias, and subsets of lymphomas. This study aims to understand the potential efficacy of epigenetic drugs called DNA methyltransferease inhibitors (DNMTi) in the treatment of leiomyosarcomas. Methods: DNMTi used in this study include azacytidine (Aza), decitabine (Dac), and guadecitabine. Aza and Dac are antimetabolites, which get incorporated onto RNA and DNA, respectively, leading to inhibition of DNMT. Guadecitabine is a novel prodrug for Dac with a longer half-life. Various leiomyosarcoma cell lines (SK-UT1, SK-LMS, and MES) were treated with varying doses of guadecitabine (0.01-5 ug/ml), decitabine (Dac: 0.1 uM-5uM), or azacitidine (Aza: 0.1 uM-5 uM) and incubated for a maximum of five days. Cell viability was measured daily via MTT assay. To further characterize the mechanism(s) of drug induced cell death, Caspase Glo® Assay (G8090) was utilized as a measure of apoptosis by proxy of Caspase 3 and 7 activity. CellToxTM Green Cytotoxicity Assay was then employed to measure membrane integrity, thus quantifying nonspecific cell death. This would further measure the toxic effect of the demethylating drugs on leiomyosarcoma cell lines. We also studied the effect of guadecitabine (3 mg/kg, 2x/week) treatment in vivo, using subcutaneous xenografts in NOD/SCID mice. Statistical analysis was conducted using GraphPad Prism 6. Results: Decreased cell viability was observed in all three leiomyosarcoma cell lines upon exposure to demethylating agents. SK-UT1 showed the highest sensitivity to all three epigenetic drugs (IC50 values: guadecitabine-0.3 uM, Aza-2.1 uM, and Dac-2.3 uM), MES was sensitive to Aza and guadecitabine (IC50 values: Aza-3.2 uM, guadecitabine-6.14 uM, and Dac-11.77 uM), and SK-LMS was sensitive only to Aza (IC50 values: Aza-2.6 uM, guadecitabine-9.6 uM, and Dac-10.3 uM). To better understand the effect of guadecitabine and AZA on these cell lines, we measured the Caspase 3/7 activity of SK-UT1 and SK-LMS using a Promega kit as a readout for apoptosis. The Caspase activity measured in SKUT1 was remarkably higher in both drugs when compared to that of SK-LMS in presence of same drugs. The amount of necrosis measured in both cell lines in presence of guadecitabine and Aza was negligible. Considerable tumor growth inhibition was also observed after in vivo treatment with guadecitabine in both SK-UT1 and SK-LMS mice xenografts. Conclusion: The use of demethylating agents leads to substantial inhibition of tumor growth in leiomyosarcoma cell lines and xenograft models. Further studies are required to explore the potential therapeutic applications and precise mechanism of action of demethylating agents in leiomyosarcoma treatment. Funding supported by ASTEX Pharmaceuticals. Note: uM= Micromole Citation Format: Cynthia De Carvalho Fischer, Yue Hu, Jasvinder Singh, Maya Thakar, Manjusha Thakar, Nita Ahuja. Treatment with demethylating drugs inhibits tumor growth in leiomyosarcoma cell lines and xenograft models [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 A35.

Abstract A11: Arginine deprivation alters global lipid metabolism in ASS1-deficient sarcomas

Abstract Objective: Argininosuccinate Synthetase 1 (ASS1) is silenced in ~90% of sarcomas. This deficiency in the urea cycle causes sarcomas to become dependent on extracellular arginine for continued cell growth and survival, a condition named arginine auxotrophy. Arginine starvation induced by pegylated arginine deiminase (ADI-PEG20) induces dramatic alterations in global metabolism as well as an induction of autophagy. We investigated the specific changes in the lipidome of leiomyosarcoma cell lines induced by ADI-PEG20. By examining the alterations in esterified and nonesterified fatty acid metabolism we sought to understand the changes in the lipidome that are capable of being therapeutically exploited. Methods: The cell lines SKLMS1 and SKUT1 were treated with ADI-PEG20. Cell pellets were collected and 33 esterified and nonesterified fatty acids were measured using GC/MS. Pathway analysis was then performed. Cell lines were then treated with a combination of ADI-PEG20 and TOFA (an acetyl CoA carboxylase inhibitor) or etoxomir/perhexiline (beta-oxidation inhibitors) to determine synthetic lethality. Results: There were clear alterations in lipid metabolism induced by arginine deprivation. The fluctuations in lipid metabolism indicated pathways susceptible to inhibition by a variety of small-molecule inhibitors, namely that arginine deprivation sensitizes to inhibition of beta-oxidation. Cell culture experiments with ADI-PEG20 and a number of different small-molecule inhibitors demonstrated synthetic lethality upon cotreatment with ADI-PEG20 and beta-oxidation inhibitors. Conclusion: Arginine deprivation causes global changes in lipid metabolism. The changes in metabolism are capable of being targeted by small-molecule inhibitors with the result being an induction of a synthetic lethality. By understanding the changes in the lipidome induced by arginine deprivation, we are building a multiagent synthetic lethal therapy for sarcoma based on metabolism that avoids chemotherapy. Citation Format: Jeff Kremer, Bethany Prudner, Samuel Chang, Lenny Rogers, Brian Van Tine. Arginine deprivation alters global lipid metabolism in ASS1-deficient sarcomas [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 A11.

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.

Increased infiltration of M2-macrophages, T-cells and PD-L1 expression in high grade leiomyosarcomas supports immunotherapeutic strategies

ABSTRACT Background: Immunotherapy may be a rational strategy in leiomyosarcoma (LMS), a tumor known for its genomic complexity. As a prerequisite for therapeutic applications, we characterized the immune microenvironment in LMS, as well as its prognostic value. Methods: CD163+ macrophages, CD3+ T-cells, PD-L1/PD-L2 and HLA class I expression (HCA2, HC10 and β2m) were evaluated using immunohistochemistry in primary tumors (n = 75), local relapses (n = 6) and metastases (n = 19) of 87 LMS patients, as well as in benign leiomyomas (n = 7). Correlation with clinicopathological parameters and survival analyses were assessed. Effect of LMS cells on macrophage differentiation was investigated using coculture of CD14+ monocytes with LMS cell lines or their conditioned media (CM). Results: 58% and 52% of the tumors were highly infiltrated with CD163+ macrophages and T-cells, respectively, with HLA class I expression observed in almost all tumors and PD-L1 expression in 30%. PD-L2 expression was also detected in some PD-L1+ tumors. All these immune markers correlated with high tumor grade but only CD163 associated with overall survival (p = 0.003) and disease-specific survival (p = 0.041). In vitro, CD163 was upregulated in the presence of LMS cells producing M-CSF, suggesting that this tumor drives macrophages towards the M2 phenotype. Conclusion: The clinical significance of M2 macrophages, possibly induced by LMS cell-secreted factors, suggests that 2/3 of high-grade LMS patients might benefit from macrophage-targeting agents. Furthermore, PD-L1 expression together with high T-cell infiltrate and HLA class I expression in around 30% of high grade LMS reflects an active immune microenvironment potentially responsive to immune checkpoint inhibitors.

Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

Background:Leiomyosarcoma (LMS) are 15% of adult sarcomas and remain seldom curable in metastatic phase. The TAM receptors and their ligands are overexpressed or activated in multiple malignancies, including LMS.Methods:The TAM receptor and ligand expression was evaluated in LMS cell lines and 358 sarcoma samples by either gene expression or immunohistochemistry. TYRO3 and AXL were knocked down. Crizotinib and foretinib were investigated in vitro.Results:High expression of TYRO3 and AXL was detected in LMS cell lines. TYRO3 or AXL gene knockdown reduced cell proliferation/colony formation. Crizotinib and foretinib decreased TYRO3 and AXL phosphorylation, apoptosis, G2/arrest and reduced colony formation. Immunohistochemistry performed in 107 sarcomas showed higher expression of TYRO3 and GAS6 in LMS vs other sarcomas and nuclear TYRO3 only in LMS. Microarray gene expression performed in 251 sarcomas revealed significantly higher expression of TYRO3 and GAS6 in LMS than other sarcomas. Leiomyosarcoma patients with high expression of GAS6 or PROS1 present a significantly worse PFS.Conclusions:Leiomyosarcoma patients, especially those whom develop metastasis, express higher levels of TYRO3 and GAS6. Crizotinib and foretinib showed effective antitumour activity in LMS through TYRO3 and AXL deactivation indicating that clinical trials using TYRO3 and AXL inhibitors are warranted in advanced LMS.

Impact of genotypic and phenotypic differences in sarcoma models on the outcome of photochemical internalization (PCI) of bleomycin

The low curative response to current treatment regimens for most soft tissue sarcomas indicates a strong need for alternative treatment strategies and predictive markers for treatment outcome. PCI (photochemical internalization) is a novel treatment strategy to translocate drugs into cytosol that otherwise would have been degraded in lysosomes. Two highly geno-and phenotypically different uterine and vulvar leiomyosarcoma cell lines, MES-SA and SK-LMS-1, were treated with bleomycin (BLM) activated by PCI (PCIBLM). The MES-SA cells were much more sensitive to PCIBLM than the SK-LMS-1 cells and the treatment induced a 7–8 fold higher increase in DNA double-strand breaks at the same dose of light as measure by γH2AX staining. A 3-fold higher induction of apoptosis and stronger activation of Bax and p21 was also measured in the P53WT MES-SA cells, compared to the P53mut SK-LMS-1 cells. The basal formation of reactive oxygen species (ROS) was 3-fold higher in SK-LMS-1 cells than in the MES-SA cells and SK-LMS-1 cells expressed glutathione peroxidase 1 (GPx1) and more superoxide dismutase 2 (SOD2) than the MES-SA cells. Glutathione depletion with the glutathione synthetase inhibitor buthionine sulfoximine increased the cytotoxic effect of the photochemical treatment (PDT) most strongly in the SK-LMS-1 cells, and reduced PCIBLM-induced H2AX activation in the MES-SA cells, but not in the SK-LMS-1 cells. The results indicate PCIBLM as a potential novel treatment strategy for soft tissue sarcomas, with antioxidant enzymes, in particular GPx1, and the P53 status as potential predictive markers for response to PCIBLM.

Establishment and proteomic characterization of NCC-LMS1-C1, a novel cell line of primary leiomyosarcoma of the bone

Leiomyosarcoma (LMS) is one of most aggressive mesenchymal malignancies that differentiate towards smooth muscle. The clinical outcome of LMS patients is poor; as such, there is an urgent need for novel therapeutic approaches. Experimental models such as patient-derived cell lines are invaluable tools for pre-clinical studies. In the present study, we established a stable cell line from the tumor tissue of a patient with a primary LMS of the bone. Despite the urgent need for novel therapeutic strategies in LMS, there are only a few LMS cell lines available in public cell banks, none of which are primary to the bone. Bone primary LMS tumor tissues were sampled to establish cell lines. Morphological and proteomic analyses were performed and sensitivity to pazopanib was evaluated. NCC-LMS1-C1 cells were maintained for over 100 passages. The cells exhibited a spindle shape and aggressive growth; they also expressed smooth muscle actin, reflecting the original LMS tissue (i.e. smooth muscle cells). The cells also showed tumor characteristics such as colony formation on soft agar and sensitivity to pazopanib, doxorubicin and cisplatin, with half-maximal inhibitory concentrations of 4.5, 0.11 and 20 μM, respectively. Proteomic analyses by mass spectrometry and antibody array revealed some differences in the protein expression profiles of these cells as compared to the original tumor tissue. Our results indicate that the NCC-LMS1-C1 cell lines will be useful for LMS research.

Abstract 1200: Novel strategy to overcome platinum resistance in uterine leiomyosarcoma; blocking ATP7B by copper ion

Abstract Objective: Resistance to platinum drugs remains a significant problem. Our objective is to elucidate the role of ATP7B, a copper transporter which excretes intracellular platinum giving drug resistance, in gynecologic malignancies and to develop a method which can overcome the platinum resistance. Methods: The expression of ATP7B was evaluated in 3 uterine leiomyosarcoma (LMS) cell lines (SK-LMS, SK-UT1 and SKN) and 26 clinical samples of LMS by immunohistochemical (IHC) staining, respectively. The IC50 values of cisplatin were measured by modified MTT assay. Intracellular platinum accumulation was determined by ICP-MS analysis in SK-LMS cells and ATP7B-stably suppressed cells (SK-LMS-7B cells), which transfected the pRS ATP7B shRNA vector. To analyze in vivo platinum sensitivity, SK-LMS and SK-LMS-7B cells were subcutaneously injected into nude mice. Cisplatin (3 mg/kg) or PBS was administered i.p. to xenografted mice twice weekly for 28 days. Further, we attempted to show the improvement of platinum sensitivity by using premedication for CuSO4 before cisplatin treatment in vitro and in vivo model. Results: Expression of ATP7B was observed in all 3 LMS cell lines. In clinical samples, ATP7B was over expressed in 14 of 26 (53.8%) cases. In drug resistance analysis, significantly lower IC50 values of cisplatin were observed in SK-LMS-7B cells (4.3μM, p < 0.01) compared with SK-LMS cells (17μM) indicating knockdown of ATP7B improved platinum sensitivity. Also, significantly higher platinum accumulation was detected in SK-LMS-7B cells (7.2ng/dish, p < 0.01) compared with SK-LMS cells (52ng/dish). In in vivo analyses, a significantly higher anti-tumor effect of cisplatin was observed in SK-LMS-7B-xenografted mice compared with SK-LMS-xenografted mice (56±27 μg vs 214±68 μg, p<0.05). Pretreatment with CuSO4 (15μM) before cisplatin treatment for SK-LMS cells significantly reduced IC50-values (17 μM to 3.9 μM, p<0.05). In addition, an elevated intracellular platinum accumulation was observed in cells from pretreatment group (15.0 to 68.4 ng/dish, p<0.05). A significant improvement of platinum sensitivity was observed in the premedication group (CuSO4 premedication group; 65±40 μg vs no pretreatment group; 230±60 μg, p<0.05). Conclusion: ATP7B contributes platinum resistance in LMS. Presumably, ATB7B preferentially excrete copper ion thereby platinum can be accumulate in cells. Thus, pretreatment with CuO4 effectively improved platinum sensitivity in vitro and in vivo by blocking ATP7B. Citation Format: Mamoru Kakuda, Shinya Matsuzaki, Ruriko Nakae, Yusuke Tanaka, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Tadashi Kimura. Novel strategy to overcome platinum resistance in uterine leiomyosarcoma; blocking ATP7B by copper ion [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 1200. doi:10.1158/1538-7445.AM2017-1200

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.

An Electrostatic Field on a Bioceramic Growth Surface Suppressed Leiomyosarcoma Cell Proliferation but Accelerated Differentiation

Living bodies make use of endogenous electric energies for biological processes and can be affected by exogenous energies as well. Certain exogenous energies, such as the electric field of a polarized bioceramic surface, have been reported to have potency as part of an adjunct therapy for malignant tumors. Here, we investigated the effects of an electrostatic field on a leiomyosarcoma cell line cultured on polarized hydroxyapatite. We found that growth on the polarized surface suppressed cell proliferation, but the occurrence of apoptosis was low. In addition, our examination of cell cyclerelated genes revealed that the expression levels of two markers of myogenic differentiation were increased and tumor suppressor genes were up-regulated. These results indicated that inhibition of DNA replication led to the suppression of proliferation and acceleration of differentiation. Further studies on the electric fields produced around polarized bioceramic surfaces and their effects on cell cycle regulation might enable the design of an effective differentiation-inducing therapy for leiomyosarcoma and other cancers.

Pioglitazone induces cell growth arrest and activates mitochondrial apoptosis in human uterine leiomyosarcoma cells by a peroxisome proliferator-activated receptor γ-independent mechanism.

The peroxisome proliferator-activated receptor γ (PPARγ) agonists, thiazolidinediones, including pioglitazone (PIO) exhibit anti-tumour activities in cancer cells. The present study investigates the effects of PIO on cell proliferation and apoptosis in SK-UT-1 cells, a human uterine leiomyosarcoma cell line, and human uterine smooth muscle cells (HUtSMC). The proliferation and viability of SK-UT-1 cells treated with vehicle or PIO were assessed by cell counting and WST-1 assay. The activity of MEK/ERK and p38 MAPK signalling pathways and the expression of p53, the cyclin-dependent kinase inhibitor, p21, Bax, Bad and Bim proteins and cleaved caspase-3 were analysed by Western blotting. Quiescent SK-UT-1 cells intensively proliferate and display high levels of phosphorylated, activated MEK1/2, ERK1/2 and p38 MAPK. PIO (10 or 25μM) induced time- and dose-dependently cell-growth arrest, reduced the cell numbers and effectively suppressed the over-activated MEK/ERK and p38 MAPK signalling pathways as evidenced by the abolished levels of phosphorylated MEK1/2, ERK1/2 and p38 MAPK. PIO activated the intrinsic apoptotic pathway, i.e. up-regulated the p53, p21, Bax and Bad proteins and cleaved caspase-3. PIO also reduced cell numbers of highly proliferative SK-UT-1 cells cultured in growth medium. The anti-proliferative and pro-apoptotic actions of PIO were not PPARγ dependent and exclusive for SK-UT-1 cells as PIO did not interfere with the proliferation of HUtSMC. The pronounced anti-tumorigenic effects of PIO in SK-UT-1 cells address an important issue about the relevance of the PPARγ agonist in the treatment of the human uterine leiomyosarcoma.

Abstract 286: Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma

Abstract Objective Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastasic 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 pan-HDACi alone and in combination with gemcitabine in LMS. Methods Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included a pan-HDAC inhibitor (HDACi) and nucleoside analog, gemcitabine (Gem). MTS and clonogenic assays were used to evaluate the effect of HDACi on LMS cell growth. Cell cycle FACS and annexin V PI/FACS analysis were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. Compusyn software was used to determine in vitro synergy studies for the combination of HDACi + Gem. A LMS xenograft model in SCID mice was used to test the impact of HDACi alone, Gem alone and the combination of HDACi + Gemcitabine. Results Pan-HDAC inhibition abrogated LMS cell growth and clonogenic potential. HDACi induced G2/M cell cycle growth arrest and enhanced apoptosis in LMS cell lines. The combination of HDACi + Gem exhibited a synergistic effect in LMS cells in vitro. Similarly, HDACi combined with Gem resulted in superior anti-LMS effects in vivo. Conclusion LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of pan-HDAC inhibition combined with gemcitabine for the treatment of LMS. Citation Format: Gonzalo Lopez, Edwin Choy, Shreyaskumar Patel, Hemant Bid, Danielle Braggio, Dina Lev, Raphael Pollock. Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma. [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 286.

Abstract 2153: ATP7B is a promising therapeutic target for uterine leiomyosarcoma

Abstract Objective Resistance to platinum drugs remains a significant problem in uterine leiomyosarcoma (LMS). We investigated the role of ATP7B in the resistance to platinum drugs in LMS using both in vitro and in vivo models. Methods The expression of the typical platinum transporters (MDR1, MRP2, ATP7A, and ATP7B) was examined in LMS cell lines using Western blotting analysis. ATP7A expression was investigated by immunohistochemistry (IHC) using clinical samples of LMS. IC50 values to cisplatin were measured in SK-LMS cells, SK-LMS-ATP7B-suppressed cell line (SK-LMS-7B cells), which permanently transfected PRS ATP7B shRNA vector. We established xenografted mice by inoculating SK-LMS cells and SK-LMS-7B cells, and examined in vivo platinum sensitivity with cisplatin for both tumors. This study was approved by the Institutional Review Board and the Ethics Committee of the Osaka University Hospital (approval #10302, approved on March 11, 2011). Results The expression of ATP7B was identified in the SK-LMS cells. ATP7B expression was identified in 70% (14/20) of the LMS clinical samples using IHC. The IC50-values to cisplatin improved from 17 mM to 4.3 mM after the suppression of ATP7B in SK-LMS-7B cells. A significant anti-tumor effect of cisplatin was observed in SK-LMS-7B xenografted mice than in SK-LMS xenografted mice. We also identified CuSO4 as an preferentially inhibitor of ATP7B in vitro. Conclusion ATP7B is associated with platinum resistance. CuSO4 acting as an inhibitor of ATP7B can be a therapeutic target for LMS. Citation Format: Mamoru Kakuda. ATP7B is a promising therapeutic target for uterine leiomyosarcoma. [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 2153.

Expression of Forkhead box M1 in soft tissue leiomyosarcoma: Clinicopathologic and invitro study using a newly established cell line.

Leiomyosarcoma (LMS) of soft tissue is a sarcoma with smooth-muscle differentiation, and conventional chemotherapy does not improve its outcome. The application of novel antitumor agents and precise prognostication has been demanded. The expression of the protein Forkhead box M1 (FOXM1), a member of the FOX family, is considered an independent predictor of poor survival in many cancers and sarcomas. However, the expression status of FOXM1 in LMS is poorly understood. The purposes of this study were to examine the correlation between the expression of FOXM1 and clinicopathologic or prognostic factors and to clarify the efficacy of FOXM1 target therapy in LMS. We evaluated the immunohistochemical expressions of FOXM1 using 123 LMS tumor specimens. Univariate and multivariate survival analyses revealed that FOXM1 expression was associated with poor prognosis in LMS. An invitro study was then carried out to examine the antitumor effect of a FOXM1 inhibitor (thiostrepton) and siRNA on a novel LMS cell line, TC616. We also assessed the efficacy of the combined use of doxorubicin and thiostrepton. Thiostrepton showed dose-dependent antitumor activity and TC616 cells treated with the combination of thiostrepton and doxorubicin showed lower proliferation compared to those treated with either drug individually. FOXM1 interruption by siRNA decreased cell proliferation and increased chemosensitivity. In conclusion, FOXM1 has potential to be a therapeutic target for LMS.

Verticillin A Inhibits Leiomyosarcoma and Malignant Peripheral Nerve Sheath Tumor Growth via Induction of Apoptosis.

ObjectiveThe heterogeneity of soft tissue sarcoma (STS) represents a major challenge for the development of effective therapeutics. Comprised of over 50 different histology subtypes of various etiologies, STS subsets are further characterized as either karyotypically simple or complex. Due to the number of genetic anomalies associated with genetically complex STS, development of therapies demonstrating potency against this STS cluster is especially challenging and yet greatly needed. Verticillin A is a small molecule natural product with demonstrated anticancer activity; however, the efficacy of this agent has never been evaluated in STS. Therefore, the goal of this study was to explore verticillin A as a potential STS therapeutic.MethodsWe performed survival (MTS) and clonogenic analyses to measure the impact of this agent on the viability and colony formation capability of karyotypically complex STS cell lines: malignant peripheral nerve sheath tumor (MPNST) and leiomyosarcoma (LMS). The in vitro effects of verticillin A on apoptosis were investigated through annexin V/PI flow cytometry analysis and by measuring fluorescently-labeled cleaved caspase 3/7 activity. The impact on cell cycle progression was assessed via cytometric measurement of propidium iodide intercalation. In vivo studies were performed using MPNST xenograft models. Tumors were processed and analyzed using immunohistochemistry (IHC) for verticillin A effects on growth (Ki67) and apoptosis (cleaved caspase 3).ResultsTreatment with verticillin A resulted in decreased STS growth and an increase in apoptotic levels after 24 h. 100 nM verticillin A induced significant cellular growth abrogation after 24 h (96.7, 88.7, 72.7, 57, and 39.7% reduction in LMS1, S462, ST88, SKLMS1, and MPNST724, respectively). We observed no arrest in cell cycle, elevated annexin, and a nearly two-fold increase in cleaved caspase 3/7 activity in all MPNST and LMS cell lines. Control normal human Schwann (HSC) and aortic smooth muscle (HASMC) cells displayed higher tolerance to verticillin A treatment compared to sarcoma cell lines, although toxicity was seen in HSC at the highest treatment dose. In vivo studies mirrored the in vitro results: by day 11, tumor size was significantly reduced in MPNST724 xenograft models with treatment of 0.25 and 0.5 mg/kg verticillin A. Additionally, IHC assessment of tumors demonstrated increased cleaved caspase 3 and decreased proliferation (Ki67) following treatment with verticillin A.ConclusionAdvancement in the treatment of karyotypically complex STS is confounded by the high level of genetic abnormalities found in these diseases. Consequently, the identification and investigation of novel therapies is greatly needed. Our data suggest that verticillin A selectively inhibits MPNST and LMS growth via induction of apoptosis while exhibiting minimal to moderate effects on normal cells, pointing to verticillin A as a potential treatment for MPNST and LMS, after additional preclinical validation.

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.

Abstract A24: Cancer-testis antigen expression is shared among gynecological tumors

Abstract Objective: Immunotherapy has become one of the novel therapeutic strategies under investigation for several cancers including ovarian cancer. Although antigenic targets for the immune system exist in the form of tumor-associated antigens, no tumor-specific antigens have been identified for gynecological cancers. Expression of cancer-testis (CT) antigens in ovarian cancer has been described. CT antigens are attractive targets for cancer immunotherapy due to their restricted expression in testis, germ cells and tumors. Current data from gene expression profiles in many types of tumors suggests that antigen sharing between allogeneic tumors may be a generalizable phenomenon. We hypothesize that there will be antigen sharing between gynecological cancers. The aim of our study was to evaluate the expression of a panel of 20 CT antigens in gynecological tumor cell lines, and to determine if antigen sharing occurs among different gynecologic cancers. Methods: RNA was isolated from 16 gynecological cancer cell lines (9 epithelial ovarian cancer, 8 endometrial cancer, 1 cervical cancer and 1 leiomyosarcoma cell line). Real time-PCR analysis was performed to determine the expression level of 20 CT antigens, including NY-ESO-1, MAGE-A3/A4, GAGE, LAGE, PIWIL-2, CT-45. Results: Expression of CT antigens was identified in gynecologic cancer cell lines. Among 20 evaluated antigens 90% (18/20) expressed in ovarian and endometrial cancer cell lines, while only 30% (6/20) expressed in cervical cancer and leyomyosarcoma cell lines. The most frequently expressed CT antigens were SP-17(93.8% of cell lines), MAGE-A4 (87.5%), OYTES (68.8%), BAGE (62.5%),CT-45 (62.5%), PIWIL-2 (56.3%) and GAGE (56.3%). CT45, SP-17 and MAGE-A4 were expressed among all gynecological cancers. We found that different gynecological cancers shared the expression of CT antigens, with the mean number of antigens shared among cancer types ranging from 3 to 4.6 antigens. There was sharing of CT antigens between cell lines of the same tumor type and with cell lines of other gynecological tumor types. The mean number of antigens shared among endometrial cell lines (5.5) was higher than the one shared by the ovarian cancer cell lines (3.8) with their corresponding tumor type (p-value= 0.0122). Conclusion: Cancer-testis antigens are expressed in gynecological cancers. No single antigen was noted universally expressed among all gynecological cancer cell lines, however most expressed SP-17. Utilizing a multiepitope immunotherapy approach could provide a universal therapeutic option for gynecological cancers. Citation Format: Arlene E. Garcia-Soto, Tulay Koru-Sengul, Feng Miao, Taylor Schreiber, Wilberto Nieves-Neira, Eckhard Podack. Cancer-testis antigen expression is shared among gynecological tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A24.

LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells

Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without β-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

Abstract 2707: Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3

Abstract The MDM2-p53 pathway plays a prominent role in various cancer pathogenesis. Nutlin-3, a small-molecule antagonist of MDM2, inhibits proliferation in cancer cells with wild-type p53 by blocking the interaction of MDM2 and p53. However, different cancer cells differ greatly sensitivity of Nutlin-3 because of MDM2 amplification and/or p53 mutation status. Herein, we evaluate the sensitivity of Nutlin-3 in different cancer cell lines including four mesothelioma cell lines, one breast cancer line, and one leiomyosarcoma cell line with nonmutant p53 expression, two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one control mesothelioma cell line JMN1B harboring p53 point mutation. Unexpectedly, Nutlin-3 treatment induced cyclin D1 expression accompanied with inhibition of MDM2-p53 interaction in nonmutant cancer cell lines. Additive effects were obtained through a coordinated attack on MDM2-p53 interaction and cyclin D1, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that lentiviral mediated cyclin D1 knockdown or drug inhibition, and block of MDM2-p53 interaction, in cancer cell lines containing wild-type p53, induced greater anti-proliferative effects, compared to either intervention alone. Further results demonstrated that upregulation of cyclin D1 after Nutlin-3 treatment to some extent was dependent on expression and ubiquitin E3-ligase activity of MDM2. These compelling anti-proliferative responses indicate that combination inhibition of cyclin D1 and MDM2-p53 interaction warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53. Citation Format: Wenbin Ou, Weicai Chen, Jiaqing Zhu, Grant Eilers, Xuhui Li, Peipei Yang, Hailong Li, Fanguo Meng, Jonathan Fletcher. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2707. doi:10.1158/1538-7445.AM2015-2707