Clinical activity of mTOR inhibition with sirolimus in malignant perivascular epithelioid cell tumors: targeting the pathogenic activation of mTORC1 in tumors.

PURPOSE Perivascular epithelioid cell tumors (PEComas) represent a family of mesenchymal neoplasms, mechanistically linked through activation of the mTOR signaling pathway. There is no known effective therapy for PEComa, and the molecular pathophysiology of aberrant mTOR signaling provided us with a scientific rationale to target this pathway therapeutically. On this mechanistic basis, we treated three consecutive patients with metastatic PEComa with an oral mTOR inhibitor, sirolimus. PATIENTS AND METHODS Patients with advanced PEComa were treated with sirolimus and consented to retrospective collection of data from their medical records and analysis of archival tumor specimens. Tumor response was determined by computed tomography scans obtained at the clinical discretion of the treating physicians. Tumors were assessed for immunohistochemical evidence of mTORC1 activation and genetic evidence of alterations in TSC1 and TSC2. Results Radiographic responses to sirolimus were observed in all patients. PEComas demonstrated loss of TSC2 protein expression and evidence of baseline mTORC1 activation. Homozygous loss of TSC1 was identified in one PEComa. CONCLUSION Inhibition of mTORC1, pathologically activated by loss of the TSC1/TSC2 tumor suppressor complex, is a rational mechanistic target for therapy in PEComas. The clinical activity of sirolimus in PEComa additionally strengthens the pathobiologic similarities linking PEComas to other neoplasms related to the tuberous sclerosis complex.

Mammalian target of rapamycin regulates murine and human cell differentiation through STAT3/p63/Jagged/Notch cascade.

The receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) pathway is frequently altered in cancer, but the underlying mechanism leading to tumorigenesis by activated mTOR remains less clear. Here we show that mTOR is a positive regulator of Notch signaling in mouse and human cells, acting through induction of the STAT3/p63/Jagged signaling cascade. Furthermore, in response to differential cues from mTOR, we found that Notch served as a molecular switch to shift the balance between cell proliferation and differentiation. We determined that hyperactive mTOR signaling impaired cell differentiation of murine embryonic fibroblasts via potentiation of Notch signaling. Elevated mTOR signaling strongly correlated with enhanced Notch signaling in poorly differentiated but not in well-differentiated human breast cancers. Both human lung lymphangioleiomyomatosis (LAM) and mouse kidney tumors with hyperactive mTOR due to tumor suppressor TSC1 or TSC2 deficiency exhibited enhanced STAT3/p63/Notch signaling. Furthermore, tumorigenic potential of cells with uncontrolled mTOR signaling was suppressed by Notch inhibition. Our data therefore suggest that perturbation of cell differentiation by augmented Notch signaling might be responsible for the underdifferentiated phenotype displayed by certain tumors with an aberrantly activated RTK/PI3K/AKT/mTOR pathway. Additionally, the STAT3/p63/Notch axis may be a useful target for the treatment of cancers exhibiting hyperactive mTOR signaling.

17beta-estradiol and predegenerated nerve graft effect on hippocampal neurogenesis in adult female rats.

OBJECTIVES: The aim of present work was to examine estrogen influence on neurogenesis in the model of predegenerated peripheral nerve grafts implantation into the rat hippocampal dentate gyrus. METHODS: Experiment was carried out on female rats divided into three experimental groups: NO - non-ovariectomized, OV - ovariectomized and E - heterogeneous group with various 17-beta-estradiol substitution after ovariectomy. Proliferating cells were labeled with BrdU. Brains were subjected to immunohistochemical procedures to visualize nestin, GFAP and estrogen receptors (ERalpha and ERbeta). RESULTS: Proliferation rate was highest in E groups with estrogen levels resembling that in proestrus phase. Ovariectomy resulted in higher than in NO group number of new neurons, while high hyperestrogenemia worsened the results. The proportions of nestin-labeled cells correlated in similar way with different hormonal state. We found also distinct co-localization of nestin and GFAP in E group (proestrus). It may suggest the presence of radial glia, a potential source of new neurons in adult mammals. Nerve graft induced ERalpha expression at the site of injury in all groups. Distribution of ERbeta in hippocampus was estradiol-dose-dependent and correlated with cell proliferation. CONCLUSION: In our model, 17-beta-estradiol and predegenerated nerve graft implantation had synergistic effect on hippocampal neurogenesis.

Renal and liver tumors in Tsc2(+/-) mice, a model of tuberous sclerosis complex, do not respond to treatment with atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor.

Inactivating mutations of the tumor suppressor gene TSC2 are associated with tumorigenesis in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Statins, as 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have the potential to limit the growth of these tumors by limiting the isoprenylation of activated GTPases in Tsc2-null cells. We tested atorvastatin as a therapy for (a) ethylnitrosourea (ENU)-enhanced renal cystadenoma and (b) spontaneous liver hemangioma in 129Sv/Jae Tsc2(+/-) mice. ENU-treated Tsc2(+/-) mice were given atorvastatin chow (0.1%, w/w) for 1 or 3 months before sacrifice at 6 months; 129Sv/Jae Tsc2(+/-) mice were given atorvastatin chow (0.1%, w/w) for 6 months before sacrifice at 12 months. All treatment groups were compared with mice of identical genotype and strain background that were fed control chow. Pathologic analyses revealed a predominance of renal cystadenoma in ENU-treated and liver hemangioma in non-ENU-treated 129Sv/Jae Tsc2(+/-) mice. In both cohorts, serum cholesterol levels and levels of phosphorylated S6 and GTP-RhoA in healthy tissue were significantly (>50%) reduced in atorvastatin-treated mice as compared with controls. Following atorvastatin treatment, no significant reduction in tumor size, morphology, or phosphorylated S6 levels was observed for either ENU-associated renal cystadenoma or spontaneous liver hemangioma as compared with the untreated groups. In conclusion, although the marked reduction in cholesterol levels indicates that atorvastatin was effective as an 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, it did not inhibit the growth of tumors that develop in these Tsc2(+/-) models, suggesting that it is unlikely to have benefit as a single-agent therapy for TSC-associated tumors.

Paraganglioma of the cauda equina presenting with erectile and sphincter dysfunction.

Paragangliomas of the cauda equina are rare neuroepithelial tumors, usually manifesting clinically as sciatica. Here, we report a case of cauda equina paraganglioma with an unusual course in a 43-year-old man. His main complaints were erectile and sphincter dysfunction. The low back pain was initially ascribed to accidental injury. Magnetic resonance imaging revealed intradural tumor at the L2/L3 level. The patient underwent gross tumor resection, and the diagnosis of paraganglioma was based on neuropathologic examination. The symptoms completely resolved after tumor resection.

Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis.

BACKGROUND: Tuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from drug-induced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235. RESULTS: Using ENU to enhance Tsc2+- kidney tumor development, both RAD001 (10 mg/kg PO 5 d/week) and NVP-BEZ235 (45 mg/kg PO QD) had equivalent effects in suppressing tumor development during a 4 week treatment period, with a 99% reduction in tumor cell mass. Marked reduction in activation of mTORC1, induction of cell cycle arrest, and absence of apoptotic cell death was seen in mice treated with either drug. However, when either was discontinued, there was prompt recovery of tumor growth, with extensive proliferation. CONCLUSION: Both mTORC1 blockade alone and combined PI3K-mTOR blockade lead to suppression of tumor development but not tumor elimination in this TSC model.

A hypomorphic allele of Tsc2 highlights the role of TSC1/TSC2 in signaling to AKT and models mild human TSC2 alleles.

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome in which hamartomas develop in multiple organ systems. Knockout and conditional alleles of Tsc1 and Tsc2 have been previously reported. Here, we describe the generation of a novel hypomorphic allele of Tsc2 (del3), in which exon 3, encoding 37 amino acids near the N terminus of tuberin, is deleted. Embryos homozygous for the del3 allele survive until E13.5, 2 days longer than Tsc2 null embryos. Embryos die from underdevelopment of the liver, deficient hematopoiesis, aberrant vascular development and hemorrhage. Mice that are heterozygous for the del3 allele have a markedly reduced kidney tumor burden in comparison with conventional Tsc2(+/-) mice. Murine embryo fibroblast (MEF) cultures that are homozygous for the del3 allele express mutant tuberin at low levels, and show enhanced activation of mTORC1, similar to Tsc2 null MEFs. Furthermore, the mutant cells show prominent reduction in the activation of AKT. Similar findings were made in the analysis of homozygous del3 embryo lysates. Tsc2-del3 demonstrates GTPase activating protein activity comparable to that of wild-type Tsc2 in a functional assay. These findings indicate that the del3 allele is a hypomorphic allele of Tsc2 with partial function due to reduced expression, and highlight the consistency of AKT downregulation when Tsc1/Tsc2 function is reduced. Tsc2-del3 mice also serve as a model for hypomorphic TSC2 missense mutations reported in TSC patients.

The comparison of multipotential for differentiation of progenitor mesenchymal-like stem cells obtained from livers of young and old rats.

The presence of stem cells differentiating to hepatocytes and cholangiocytes has been previously reported in livers of young rats. Here, we have isolated, cultured, and characterized mesenchymal stem cells (MSCs) from livers of young and old rats and tested their multipotential for differentiation. The mesenchymal stem cells in liver sections were identified by the presence of markers, respectively for primary stem cells Thy-1 and CD34, for differentiation to early cholangiocytes GST and CK19, and for differentiation to hepatocytes GSTalpha and CK18. Ki67 was detected as the cell proliferation marker. Cells isolated from livers of either age group were tested in a culture for their viability following storage and were characterized for the presence of most of the markers detected in cells in situ. The results revealed age-dependent changes in the number of recovered primary MSCs. In both age groups we have observed cells changing under differentiating conditions to liver cell lineages, such as cholangiocytes and hepatocytes, as well as to non-liver cells such as adipocytes, astrocytes, neuroblasts, and osteoblasts. Our data revealed that from the livers of rats 20 months and older the primary MSCs could be isolated and expanded; however, they were significantly fewer, even though their differentiation multipotential was preserved. The mechanism involved in the differentiation of liver MSCs seemed to depend on a constellation of signals in Notch signalling pathways. Thus, our results support the idea of potential use of liver as a source of MSCs, not only for liver reconstruction but also for cell therapy in general.

Total ghrelin plasma level in patients with the restrictive type of anorexia nervosa.

Ghrelin is produced mainly in the stomach and is an essential link of the brain-gut axis. Ghrelin stimulates hunger centers in hypothalamus controlling food intake and body mass gain. The aim of the study is to analyze the total ghrelin plasma level in patients suffering from restrictive type of anorexia nervosa (AN-R). According to DSM-IV classification a group of 30 AN-R patients was investigated before and after 3 and 6 months of therapy. Therapy included normocaloric diet and cognitive-behavioral psychotherapy (CBT). The control group consisted of 20 girls without any eating disorders. Before the therapy the total ghrelin plasma level in AN-R patients was significantly higher than in the control group. After 3 and 6 months of treatment the total ghrelin plasma level in AN-R patients was significantly lower than in the control group. In AN-R patients, the total ghrelin plasma level is connected with the pathological feeding behavior.

Effect of oblique nerve grafting on peripheral nerve regeneration in rats.

Current methods of peripheral nerve repair are to rejoin cut nerve stumps directly or to bridge large gaps with autologous nerve grafts. In both cases the surface of nerve stump endings is typically cut perpendicularly to the long axis of the nerve. The outcome of such operations, however, is still not satisfactory. In this study, we examine the effect of oblique nerve cutting and grafting on morphological as well as functional features of regeneration. In adult rats, sciatic nerve was cut and rejoined either directly or using an autologous graft, at 90 degrees or 30 degrees angle. Functional regeneration was assessed by walking track analysis during 12-week follow-up. Afterwards muscle weight was measured and histological studies were performed. The latter included nerve fibers and Schwann cells counting, as well as visualization of scar formation and epineural fibrosis. Nerves cut obliquely and rejoined showed better functional recovery than perpendicularly transected. Similar effect was observed after oblique grafting when compared to perpendicular one. Numbers of nerve fibers growing into the distal stump of the nerve as well as the number of Schwann cells were significantly higher in obliquely than in perpendicularly operated nerves. Moreover, growing axons were arranged more regularly following oblique treatment. These data indicate that joining or grafting the nerve stumps at acute angle is a more profitable method of nerve repair than the standard procedure performed at right angle.

Expression of Bcl-2 and Bax protein in normal pineal gland in children and young adult.

The Bcl family contains both pro and antiapoptotic proteins participating in the regulation of neuronal cell death in several pathological conditions. However, very little is known about physiological profiles of Bcl-2/Bax expression in normal brain. In this study, we examined expression profile of Bcl-2 and Bax proteins in normal pineal gland in children. The material for analysis was obtained by biopsy of pineal parenchyma during surgery of pineal cysts. All specimens were labeled immunohistochemically and analyzed by means of confocal laser scanning microscope. We found only few Bcl-2 expressing (0.7%) and no Bax-immunopositive (0.0%) pinealocytes. Bcl-2-positive cells were mature neurons, neither young ones nor glia.

[How estrogens will surprise us again?]. / Czym jeszcze zaskocza nas estrogeny?

Estrogens are pleiotropic hormones having an influence not only on reproductive system and sexual functions. These hormones are synthesized not only by ovaries, but also by glia in central nervous system (CNS) and Schwann cells in peripheral nervous system. Therefore they create microenvironment having a wide spectrum of effects such as neuroprotective and antiapoptotic or supporting neurogenesis and regeneration. Mechanisms of estrogens activity are both genomic and quick non-genomic transmitted through second intracellular messengers. There is evidence for protective action of estrogens in neurodegenerative diseases and other diseases of CNS. Nevertheless there are still secrets in estrogens nature. This fact pushes us to ask more questions and continue scientific research to look for the answer.