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1.
Int J Mol Sci ; 22(10)2021 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-34065204

RESUMEN

Schwann cells are normally quiescent, myelinating glia cells of the peripheral nervous system. Their aberrant proliferation and transformation underlie the development of benign tumors (neurofibromas) as well as deadly malignant peripheral nerve sheath tumors (MPNSTs). We discovered a new driver of MPNSTs, an oncogenic GTPase named RABL6A, that functions in part by inhibiting the RB1 tumor suppressor. RB1 is a key mediator of cellular senescence, a permanent withdrawal from the cell cycle that protects against cell immortalization and transformation. Based on the RABL6A-RB1 link in MPNSTs, we explored the hypothesis that RABL6A promotes Schwann cell proliferation and abrogates their senescence by inhibiting RB1. Using sequentially passaged normal human Schwann cells (NHSCs), we found that the induction of replicative senescence was associated with reduced expression of endogenous RABL6A. Silencing RABL6A in low passage NHSCs caused premature stress-induced senescence, which was largely rescued by co-depletion of RB1. Consistent with those findings, Rabl6-deficient MEFs displayed impaired proliferation and accelerated senescence compared to wildtype MEFs. These results demonstrate that RABL6A is required for maintenance of proper Schwann cell proliferation and imply that aberrantly high RABL6A expression may facilitate malignant transformation.


Asunto(s)
Senescencia Celular/fisiología , Proteínas Oncogénicas/metabolismo , Proteínas de Unión a Retinoblastoma/metabolismo , Células de Schwann/metabolismo , Células de Schwann/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Carcinogénesis/metabolismo , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Neurofibroma/metabolismo , Neurofibrosarcoma/metabolismo
2.
bioRxiv ; 2023 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-37066322

RESUMEN

Small bowel neuroendocrine tumors (SBNETs) originate from enterochromaffin cells in the intestine which synthesize and secrete serotonin. SBNETs express high levels of tryptophan hydroxylase 1 (Tph1), a key enzyme in serotonin biosynthesis. Patients with high serotonin level may develop carcinoid syndrome, which can be treated with somatostatin analogues and the Tph1 inhibitor telotristat ethyl in severe cases. Although the active drug telotristat can efficiently reduce serotonin levels, its effect on tumor growth is unclear. This study determined the effect of serotonin inhibition on tumor cell growth in vitro and in vivo . The levels of Tph1 in various neuroendocrine neoplasms (NENs) were determined and the biological effects of Tph1 inhibition in vitro and in vivo using genetic and pharmacologic approaches was tested. Gene and protein expression analyses were performed on patient tumors and cancer cell lines. shRNAs targeting TPH1 were used to create stable knockdown in BON cells. Control and knockdown lines were assessed for their growth rates in vitro and in vivo , angiogenesis potential, serotonin levels, endothelial cell tube formation, tumor weight, and tumor vascularity. TPH1 is highly expressed in SBNETs and many cancer types. TPH1 knockdown cells and telotristat treated cells showed similar growth rates as control cells in vitro . However, TPH1 knockdown cells formed smaller tumors in vivo and tumors were less vascularized. Although Tph1 inhibition with telotristat showed no effect on tumor cell growth in vitro , Tph1 inhibition reduced tumor formation in vivo . Serotonin inhibition in combination with other therapies is a promising new avenue for targeting metabolic vulnerabilities in NENs.

3.
Clin Cancer Res ; 29(17): 3484-3497, 2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37410426

RESUMEN

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. EXPERIMENTAL DESIGN: Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. RESULTS: Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CONCLUSIONS: CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.


Asunto(s)
Neurofibrosarcoma , Ratones , Humanos , Animales , Neurofibrosarcoma/tratamiento farmacológico , Células Plasmáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasas de Proteína Quinasa Activadas por Mitógenos , Línea Celular Tumoral , Microambiente Tumoral , Quinasa 4 Dependiente de la Ciclina
4.
BMC Res Notes ; 15(1): 219, 2022 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-35752869

RESUMEN

OBJECTIVE: Inflammation is present in many diseases and identification of immune cell infiltration is a common assessment. CD138 (syndecan-1) is a recommended immunohistochemical marker for human plasmacytes although it is also expressed in various epithelia and tumors. Similarly, CD138 is a marker for murine plasmacytes, but its tissue immunostaining is not well-defined. Endogenous CD138 expression is an important confounding factor when evaluating plasmacyte infiltration. We studied two plasmacyte markers (CD138 and Kappa light chains) for endogenous immunostaining in five organs and one tumor from B6 mice. RESULTS: Plasmacytes in Peyer's patches were positive for CD138 and Kappa markers without endogenous immunostaining. Endogenous CD138 immunostaining was widespread in liver, kidney, lung and a malignant peripheral nerve sheath tumor (MPNST) versus regionalized immunostaining in skin and small intestine wall. Endogenous Kappa immunostaining was absent in all tissues except for plasmacytes. Tissues with widespread endogenous CD138 immunostaining were contrasted by absence of endogenous Kappa immunostaining. Here, plasmacytes would not be distinguished by CD138, but would be obvious by Kappa immunostaining. Our study suggests that utility of immunostaining for plasmacytes by CD138 is tissue dependent in mice. Additionally, Kappa immunostaining may be a useful alternative in mouse tissues with confounding endogenous CD138 immunostaining.


Asunto(s)
Neoplasias , Sindecano-1 , Animales , Biomarcadores/metabolismo , Inmunohistoquímica , Ratones , Neoplasias/metabolismo , Células Plasmáticas/metabolismo , Sindecano-1/metabolismo
5.
Neurooncol Adv ; 4(1): vdac047, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35571990

RESUMEN

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted along with NF1, a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of MPNSTs is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown. Methods: Using CRISPR-Cas9 targeting of Nf1 + Cdkn2a or Nf1 + Tp53 in the mouse sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Terminal tumors were evaluated by western blot, qRT-PCR, and immunohistochemistry. Results: Mice lacking Rabl6 displayed slower tumor progression and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in Rabl6-null, Nf1 + Cdkn2a lesions whereas loss of RABL6A caused upregulation of the CDK inhibitor, p27, in all tumors. Paradoxically, both models displayed elevated Myc protein and Ki67 staining in terminal tumors lacking RABL6A. In Nf1 + p53 tumors, cellular atypia and polyploidy were evident and increased by RABL6A loss. Conclusions: These findings demonstrate that RABL6A is required for optimal progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for unwanted alterations, including increased Myc, cellular atypia, and polyploidy, that ultimately promote a hyper-proliferative tumor phenotype akin to drug-resistant lesions.

6.
Sci Rep ; 11(1): 10252, 2021 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-33986468

RESUMEN

Pancreatic neuroendocrine neoplasms (pNENs) are slow growing cancers of increasing incidence that lack effective treatments once they become metastatic. Unfortunately, nearly half of pNEN patients present with metastatic liver tumors at diagnosis and current therapies fail to improve overall survival. Pre-clinical models of pNEN metastasis are needed to advance our understanding of the mechanisms driving the metastatic process and for the development of novel, targeted therapeutic interventions. To model metastatic dissemination of tumor cells, human pNEN cell lines (BON1 and Qgp1) stably expressing firefly luciferase (luc) were generated and introduced into NSG immunodeficient mice by intracardiac (IC) or intravenous (IV) injection. The efficiency, kinetics and distribution of tumor growth was evaluated weekly by non-invasive bioluminescent imaging (BLI). Tumors formed in all animals in both the IC and IV models. Bioluminescent Qgp1.luc cells preferentially metastasized to the liver regardless of delivery route, mimicking the predominant site of pNEN metastasis in patients. By comparison, BON1.luc cells most commonly formed lung tumors following either IV or IC administration and colonized a wider variety of tissues than Qgp1.luc cells. These models provide a unique platform for testing candidate metastasis genes and anti-metastatic therapies for pNENs.


Asunto(s)
Mediciones Luminiscentes/métodos , Metástasis de la Neoplasia/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Metástasis Linfática , Ratones , Ratones Endogámicos NOD , Metástasis de la Neoplasia/fisiopatología , Trasplante de Neoplasias , Neoplasias Primarias Secundarias , Células Neuroendocrinas/metabolismo , Células Neuroendocrinas/patología , Neoplasias Pancreáticas/fisiopatología
7.
Biomedicines ; 9(6)2021 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-34199469

RESUMEN

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet ß cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

8.
Clin Cancer Res ; 26(12): 2997-3011, 2020 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-32086342

RESUMEN

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs. EXPERIMENTAL DESIGN: We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors. RESULTS: RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G1 arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in vitro in an RABL6A-dependent manner and suppressed MPNST growth in vivo. Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation. CONCLUSIONS: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.


Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos , Neurofibrosarcoma/tratamiento farmacológico , Proteínas Oncogénicas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neurofibrosarcoma/genética , Neurofibrosarcoma/metabolismo , Neurofibrosarcoma/patología , Proteínas Oncogénicas/genética , Proteínas de Unión a Retinoblastoma/genética , Transducción de Señal , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP rab/genética
9.
Clin Cancer Res ; 26(8): 2011-2021, 2020 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-31937620

RESUMEN

PURPOSE: Pancreatic neuroendocrine tumors (pNETs) are uncommon malignancies noted for their propensity to metastasize and comparatively favorable prognosis. Although both the treatment options and clinical outcomes have improved in the past decades, most patients will die of metastatic disease. New systemic therapies are needed. EXPERIMENTAL DESIGN: Tissues were obtained from 43 patients with well-differentiated pNETs undergoing surgery. Gene expression was compared between primary tumors versus liver and lymph node metastases using RNA-Seq. Genes that were selectively elevated at only one metastatic site were filtered out to reduce tissue-specific effects. Ingenuity pathway analysis (IPA) and the Connectivity Map (CMap) identified drugs likely to antagonize metastasis-specific targets. The biological activity of top identified agents was tested in vitro using two pNET cell lines (BON-1 and QGP-1). RESULTS: A total of 902 genes were differentially expressed in pNET metastases compared with primary tumors, 626 of which remained in the common metastatic profile after filtering. Analysis with IPA and CMap revealed altered activity of factors involved in survival and proliferation, and identified drugs targeting those pathways, including inhibitors of mTOR, PI3K, MEK, TOP2A, protein kinase C, NF-kB, cyclin-dependent kinase, and histone deacetylase. Inhibitors of MEK and TOP2A were consistently the most active compounds. CONCLUSIONS: We employed a complementary bioinformatics approach to identify novel therapeutics for pNETs by analyzing gene expression in metastatic tumors. The potential utility of these drugs was confirmed by in vitro cytotoxicity assays, suggesting drugs targeting MEK and TOP2A may be highly efficacious against metastatic pNETs. This is a promising strategy for discovering more effective treatments for patients with pNETs.


Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Evaluación Preclínica de Medicamentos/métodos , Regulación Neoplásica de la Expresión Génica , Terapia Molecular Dirigida , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Adulto , Anciano , Línea Celular Tumoral , Biología Computacional/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Pronóstico , RNA-Seq/métodos
10.
J Clin Invest ; 129(4): 1641-1653, 2019 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-30721156

RESUMEN

Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.


Asunto(s)
Carcinoma Neuroendocrino/enzimología , Proteínas Oncogénicas/metabolismo , Neoplasias Pancreáticas/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/patología , Línea Celular Tumoral , Activadores de Enzimas/farmacología , Fase G1/efectos de los fármacos , Fase G1/genética , Humanos , Proteínas Oncogénicas/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas de Unión al GTP rab/genética
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