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Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner.

ABSTRACT: Few therapeutic options are available for malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1 (NF1). Guided by clinical observations suggesting that some NF1-associated nerve sheath tumors are hormonally responsive, we hypothesized that the selective estrogen receptor (ER) modulator tamoxifen would inhibit MPNST tumorigenesis in vitro and in vivo. To test this hypothesis, we examined tamoxifen effects on MPNST cell proliferation and survival, MPNST xenograft growth, and the mechanism by which tamoxifen impeded these processes. We found that 1-5 ?M 4-hydroxy-tamoxifen induced MPNST cell death, whereas 0.01-0.1 ?M 4-hydroxy-tamoxifen inhibited mitogenesis. Dermal and plexiform neurofibromas, MPNSTs, and MPNST cell lines expressed ER? and G-protein-coupled ER-1 (GPER); MPNSTs also expressed estrogen biosynthetic enzymes. However, MPNST cells did not secrete 17?-estradiol, exogenous 17?-estradiol did not stimulate mitogenesis or rescue 4-hydroxy-tamoxifen effects on MPNST cells, and the steroidal antiestrogen ICI-182,780 did not mimic tamoxifen effects on MPNST cells. Further, ablation of ER? and GPER had no effect on MPNST proliferation, survival, or tamoxifen sensitivity, indicating that tamoxifen acts via an ER-independent mechanism. Consistent with this hypothesis, inhibitors of calmodulin (trifluoperazine, W-7), another known tamoxifen target, recapitulated 4-hydroxy-tamoxifen effects on MPNST cells. Tamoxifen was also effective in vivo, demonstrating potent antitumor activity in mice orthotopically xenografted with human MPNST cells. We conclude that 4-hydroxy-tamoxifen inhibits MPNST cell proliferation and survival via an ER-independent mechanism. The in vivo effectiveness of tamoxifen provides a rationale for clinical trials in cases of MPNSTs.

SUBMITTER: Byer SJ

PROVIDER: S-EPMC3018903 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner.

Byer Stephanie J SJ Eckert Jenell M JM Brossier Nicole M NM Clodfelder-Miller Buffie J BJ Turk Amy N AN Carroll Andrew J AJ Kappes John C JC Zinn Kurt R KR Prasain Jeevan K JK Carroll Steven L SL

Neuro-oncology 20101112 1

Few therapeutic options are available for malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1 (NF1). Guided by clinical observations suggesting that some NF1-associated nerve sheath tumors are hormonally responsive, we hypothesized that the selective estrogen receptor (ER) modulator tamoxifen would inhibit MPNST tumorigenesis in vitro and in vivo. To test this hypothesis, we examined tamoxifen effects on MPNST cell proliferation ...[more]

PMID: 21075781

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Project description:BackgroundWe have found that erbB receptor tyrosine kinases drive Ras hyperactivation and growth in NF1-null malignant peripheral nerve sheath tumors (MPNSTs). However, MPNSTs variably express multiple erbB receptors with distinct functional characteristics and it is not clear which of these receptors drive MPNST pathogenesis. Here, we test the hypothesis that altered erbB4 expression promotes MPNST pathogenesis by uniquely activating key cytoplasmic signaling cascades.MethodsErbB4 expression was assessed using immunohistochemistry, immunocytochemistry, immunoblotting and real-time PCR. To define erbB4 functions, we generated mice that develop MPNSTs with floxed Erbb4 alleles (P0-GGFβ3;Trp53+/-;Erbb4flox/flox mice) and ablated Erbb4 in these tumors. MPNST cell proliferation and survival was assessed using 3H-thymidine incorporation, MTT assays, Real-Time Glo and cell count assays. Control and Erbb4-null MPNST cells were orthotopically xenografted in immunodeficient mice and the growth, proliferation (Ki67 labeling), apoptosis (TUNEL labeling) and angiogenesis of these grafts was analyzed. Antibody arrays querying cytoplasmic kinases were used to identify erbB4-responsive kinases. Pharmacologic or genetic inhibition was used to identify erbB4-responsive kinases that drive proliferation.ResultsAberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P0-GGFβ3 and P0-GGFβ3;Trp53+/- mice); multiple erbB4 splice variants that differ in their ability to activate PI3 kinase and nuclear signaling were present in MPNST-derived cell lines. Erbb4-null MPNST cells demonstrated decreased proliferation and survival and altered morphology relative to non-ablated controls. Orthotopic allografts of Erbb4-null cells were significantly smaller than controls, with reduced proliferation, survival and vascularization. ERBB4 knockdown in human MPNST cells similarly inhibited DNA synthesis and viability. Although we have previously shown that broad-spectrum erbB inhibitors inhibit Ras activation, Erbb4 ablation did not affect Ras activation, suggesting that erbB4 drives neoplasia via non-Ras dependent pathways. An analysis of 43 candidate kinases identified multiple NRG1β-responsive and erbB4-dependent signaling cascades including the PI3K, WNK1, STAT3, STAT5 and phospholipase-Cγ pathways. Although WNK1 inhibition did not alter proliferation, inhibition of STAT3, STAT5 and phospholipase-Cγ markedly reduced proliferation.ConclusionsErbB4 promotes MPNST growth by activating key non-Ras dependent signaling cascades including the STAT3, STAT5 and phospholipase-Cγ pathways. ErbB4 and its effector pathways are thus potentially useful therapeutic targets in MPNSTs.

Project description:The 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. We 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). Treatment 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. Advancement 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.

Project description:Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. In addition to skin pigmentation and cutaneous neurofibroma, some patients developed the plexiform neurofibroma since birth. Plexiform neurofibroma has abundant Schwann cells, fibroblasts, mast cells, blood vessels, and connective tissues, which increases the risk of developing a malignant peripheral nerve sheath tumor (MPNST). MPNST is a highly invasive cancer with no effective therapeutic agent. Cordycepin or 3'-deoxyadenosine is an extract from cordyceps militaris, which has been reported as an anti-inflammation and anti-tumor agent. Herein, we evaluated cordycepin's anti-proliferative effect on MPNST cell lines both in vitro and in vivo. Cordycepin inhibited the MPNST cell growth with an arrest of cell cycle at G2/M and S phases. The administration of naringin and pentostatin, inhibitors for adenosine deaminase (ADA), enzyme responsible for cordycepin degradation, did not show a synergistic effect in MPNST cells treated with cordycepin. However, the combined treatment enhanced the decrease of tumors in xenograft mouse model. Immunoblotting showed a decreased level of p53 protein in all MPNST cell lines, but S462TY cells. After cordycepin treatment, the levels of ERK, survivin, pAKT, and Sp1 proteins also decreased. The level of tubulin, but not actin or GAPDH, decreased in a dose-dependent manner. The microtubule network which is composed of tubulins was markedly decomposed in those treated MPNST cells. To elucidate the epigenetic control of transcription, ChIP-qPCR assay of the Sp1 and tubulin promoter regions revealed decreased Sp1 binding. The incorporation of 3'-doexyadenosine is detrimental for the process of poly(A) tail elongation. The poly(A) tail length assay showed the tail length in Sp1 and tubulin transcripts decreased in the treated cells. Nevertheless, the administration of SP1 protein to the treated cells could not rescue them completely. Furthermore, the p53-knocked-down cells (S462TY) where the expression of both p53 and Sp1 was suppressed, were vulnerable to cordycepin. The p53 protein could ameliorate the effect. In summary, cordycepin is effective to inhibit the growth of MPNST, probably through the pathway of p53/Sp1/tubulin.

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Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner.

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Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner.

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