Ral Overactivation in Malignant Peripheral Nerve Sheath Tumors

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.The Ras family of guanine-nucleotide bound proteins exerts a fundamental role in cell biology and constitutes an important area of cancer research due to its significant involvement in the development and progression of malignancies (8, 10, 17, 18, 32). Ras-like (Ral) proteins are crucial members of this family and have been shown to play a pivotal role in human tumors (7, 28, 41, 66, 70). Because Ral guanine nucleotide exchange factors (Ral-GEFs) are direct effectors of Ras, the Ral signaling pathway has been traditionally considered a Ras-effector pathway. Activation of Ras (in resemblance to Ral) is regulated by two classes of proteins: Ras-GEFs (e.g., SOS) and Ras- GTPase activating proteins (Ras-GAPs such as neurofibromin). The latter induces hydrolysis of Ras from the active (GTP) form to the inactive (GDP) form (13). Ral-GEFs include two main groups: the proteins that are stimulated by Ras because of their carboxy-terminal Ras binding domain (RalGDS, RGL1, and RGL2) and the proteins that are activated by substrates of PI3K through a pleckstrin homology domain on their C-terminal (RALGPS1 and RALGPS2) (19). Although highly similar to Ras, Ral proteins (RalA and RalB) involve a series of distinctly different effectors that influence gene expression and translation through interaction with ZO-1-associated nucleic acid binding protein (ZONAB) and RalA binding protein 1 (RalBP1) (11, 23, 33). RalB directly interacts with the SEC5 subunit of exocyst to facilitate the host defense response (48, 58).In addition to overactivation of GEFs, inactivation of GAPs is another mechanism for overactivation of GTP-bound proteins. The lack of neurofibromin (encoded by NF1 on human chromosome 17q11.2), a Ras-GAP protein, is the main molecular event in neurofibromatosis type 1 (NF-1), an autosomal-dominant human genetic disease occurring in approximately 1 in 2,500 to 3,500 births (22, 27, 42). One of the main tumor-causing effects of inactivating mutations in the tumor suppressor NF1 gene is postulated to be the subsequent activation of Ras (3, 29, 53, 57, 69). With two main functional domains, SEC14 and Ras-GAP, neurofibromin is best known for its Ras-GAP function. Although the yeast SEC14p is shown to be involved in regulating intracellular proteins and lipid trafficking, the function of its homologous domain in neurofibromin is unknown (49, 62). Although neurofibromas are the most common tumors in NF-1, 10% of patients with plexiform develop malignant peripheral nerve sheath tumors (MPNSTs), which are typically high grade and often fatal (21, 34, 65).The molecular events involved in the malignant transformation of benign neurofibromas to MPNST are poorly defined. Usually arising in the third through sixth decades of life, these tumors are composed of tightly packed hyperchromatic spindle-shaped cells with frequent mitotic figures. Inactivation of both copies of the NF1 gene has been demonstrated in benign human neurofibromas and shown to cause tumors in murine models (56). Loss of heterozygosity of NF1 and p53 has frequently been observed in human MPNST (35, 47, 54). Recombinant mouse strains (NP mice), which harbor inactivated Nf1 and p53 alleles (cis-Nf1^+/−:p53^+/−), demonstrate the cumulative effects of loss of both Nf1 and p53 genes in the etiology of MPNST (14, 68).In the present study, we show that while both Ras activation and activation of a series of its downstream effector pathways are observed in a fraction of MPNST cells, RalA is activated globally in all studied mouse and human MPNST cells and tumor samples. Our results also explain the involvement of this signaling molecule in a series of key biological functions of MPNST cells, as shown in a variety of in vitro assays and an in vivo model of MPNST. Such information may play a role in designing novel therapies for treatment of MPNST or other tumors with overactivation of the Ral pathway.     

Impaired Pten Expression in Human Malignant Peripheral Nerve Sheath Tumours

Malignant peripheral nerve sheath tumours (MPNST) are aggressive sarcomas that develop in about 10% of patients with the genetic disease neurofibromatosis type 1 (NF1). Molecular alterations contributing to MPNST formation have only partially been resolved. Here we examined the role of Pten, a key regulator of the Pi3k/Akt/mTOR pathway, in human MPNST and benign neurofibromas. Immunohistochemistry showed that Pten expression was significantly lower in MPNST (n = 16) than in neurofibromas (n = 16) and normal nervous tissue. To elucidate potential mechanisms for Pten down-regulation or Akt/mTOR activation in MPNST we performed further experiments. Mutation analysis revealed absence of somatic mutations in PTEN (n = 31) and PIK3CA (n = 38). However, we found frequent PTEN promotor methylation in primary MPNST (11/26) and MPNST cell lines (7/8) but not in benign nerve sheath tumours. PTEN methylation was significantly associated with early metastasis. Moreover, we detected an inverse correlation of Pten-regulating miR-21 and Pten protein levels in MPNST cell lines. The examination of NF1−/− and NF1+/+Schwann cells and fibroblasts showed that Pten expression is not regulated by NF1. To determine the significance of Pten status for treatment with the mTOR inhibitor rapamycin we treated 5 MPNST cell lines with rapamycin. All cell lines were sensitive to rapamycin without a significant correlation to Pten levels. When rapamycin was combined with simvastatin a synergistic anti-proliferative effect was achieved. Taken together we show frequent loss/reduction of Pten expression in MPNST and provide evidence for the involvement of multiple Pten regulating mechanisms.     

A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP) ^ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP) ^ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants. Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP) ^ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development.     

BET Bromodomain Inhibition Triggers Apoptosis of NF1-Associated Malignant Peripheral Nerve Sheath Tumors through Bim Induction

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Diagnostic Accuracy of PET/CT-Guided Percutaneous Biopsies for Malignant Peripheral Nerve Sheath Tumors in Neurofibromatosis Type 1 Patients

Background

Malignant peripheral nerve sheath tumors (MPNST) are one of the most frequent causes of death in patients with neurofibromatosis type 1 (NF1). Early detection is crucial because complete surgical resection is the only curative treatment. It has been previously reported that an ¹⁸F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) image with a T/L (Tumor/Liver) SUV_max ratio > 1.5 provides a high negative predictive value; however, it is not specific enough to make a NF1-related MPNST diagnosis. A formal proof of malignant transformation from a histological analysis is necessary before surgical excision because the procedure can cause mutilation. The objective of the present work was to investigate the effectiveness of and complications associated with PET/CT-guided percutaneous biopsies for an NF1-related MPNST diagnosis.

Methods

PET/CT-guided percutaneous biopsy procedures performed on 26 NF1 patients with a clinical suspicion of MPNST and a suspect lesion from a PET/CT scan (T/L SUV_max ratio > 1.5) were retrospectively evaluated. The localization of the suspected malignant site was determined using PET/CT. A stereotactic (ultrasonic and CT control) core biopsy technique was used with a local anesthesia.

Results

The first PET/CT-guided percutaneous biopsies enabled a pathological diagnosis for all of the patients (no "inconclusive " results were obtained), and no secondary procedures were needed. Among the 26 patients, the histopathological results from the biopsy were malignant in 17 cases and benign (BPNST with atypical cells) in nine cases. No complications from the diagnostic procedure were observed. A surgical resection was performed in 18 patients (seven benign and 11 malignant biopsies), removing the fine needle biopsy scar. In addition, six locally advanced/metastatic MPNST were treated with chemo/radiotherapy, and two BPNST had no progression after a follow-up of 14 and 39 months, respectively. The PET/CT-guided percutaneous biopsy gave 25 accurate diagnoses and one false negative (BPNST with atypical cells on the biopsy and MPNST on the operated tumor), resulting in a diagnostic accuracy rate of 96%. This false negative case may be explained by the high heterogeneity of the tumor: benign areas were contiguous with the malignant ones and associated with inflammation.

Conclusions

PET/CT-guided percutaneous biopsies are an effective and relatively non-traumatic procedure for diagnosis of NF1-related MPNST. It is the most reliable approach for early detection of MPNST.     

Aberrant cAMP Metabolism in NF1 Malignant Peripheral Nerve Sheath Tumor Cells

Malignant peripheral nerve sheath (MPNST) cell lines derived from patients with neurofibromatosis type 1 (NF!) were found to have basal cAMP levels which are two-fold higher than cAMP levels in normal human adult Schwann cells (nHSC). PCR analysis also revealed that normal adult human Schwann cells express mRNA for types Ill, IV, and IX adenylyl cyclase (AC) while NF1 MPNST cells express AC mRNA of types II, V, and VIII in addition to expressing all the isoforms of normal adult human Schwann cells. Further PCR analysis revealed that NF1 MPNST lines express mRNA for EP2 and EP4 prostaglandin receptors whereas nHSC only express mRNA for the EP2 receptor. Exogenous prostaglandins alone or in combination with PDGF BB induced greater increases in cAMP levels and proliferation of NF1 MPNST cells compared to nHSC. We conclude that aberrant cAMP signaling in NF1 MPNST cells contributes to tumor formation in NF1 patients.     

BNIP3 Regulates AT101 [(-)-Gossypol] Induced Death in Malignant Peripheral Nerve Sheath Tumor Cells

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived sarcomas and are the leading cause of mortality in patients with neurofibromatosis type 1 (NF1). Current treatment modalities have been largely ineffective, resulting in a high rate of MPNST recurrence and poor five-year patient survival. This necessitates the exploration of alternative chemotherapeutic options for MPNST patients. This study sought to assess the cytotoxic effect of the BH3-mimetic AT101 [(-)-gossypol] on MPNST cells in vitro and to identify key regulators of AT101-induced MPNST cell death. We found that AT101 caused caspase-independent, non-apoptotic MPNST cell death, which was accompanied by autophagy and was mediated through HIF-1α induced expression of the atypical BH3-only protein BNIP3. These effects were mediated by intracellular iron chelation, a previously unreported mechanism of AT101 cytotoxicity.     

Peripheral Nerve Diffusion Tensor Imaging: Assessment of Axon and Myelin Sheath Integrity

Purpose

To investigate the potential of diffusion tensor imaging (DTI) parameters as in-vivo biomarkers of axon and myelin sheath integrity of the median nerve in the carpal tunnel as validated by correlation with electrophysiology.

Methods

MRI examinations at 3T including DTI were conducted on wrists in 30 healthy subjects. After manual segmentation of the median nerve quantitative analysis of fractional anisotropy (FA) as well as axial, radial and mean diffusivity (AD, RD, and MD) was carried out. Pairwise Pearson correlations with electrophysiological parameters comprising sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) as markers of axon integrity, and distal motor latency (dml) and sensory nerve conduction velocity (sNCV) as markers of myelin sheath integrity were computed. The significance criterion was set at P=0.05, Bonferroni corrected for multiple comparisons.

Results

DTI parameters showed a distinct proximal-to-distal profile with FA, MD, and RD extrema coinciding in the center of the carpal tunnel. AD correlated with CMAP (r=0.50, p=0.04, Bonf. corr.) but not with markers of myelin sheath integrity. RD correlated with sNCV (r=-0.53, p=0.02, Bonf. corr.) but not with markers of axon integrity. FA correlated with dml (r=-0.63, p=0.002, Bonf. corr.) and sNCV (r=0.68, p=0.001, Bonf. corr.) but not with markers of axon integrity.

Conclusion

AD reflects axon integrity, while RD (and FA) reflect myelin sheath integrity as validated by correlation with electrophysiology. DTI parameters consistently indicate a slight decrease of structural integrity in the carpal tunnel as a physiological site of median nerve entrapment. DTI is particularly sensitive, since these findings are observed in healthy participants. Our results encourage future studies to evaluate the potential of DTI in differentiating axon from myelin sheath injury in patients with manifest peripheral neuropathies.     

CD44V6和MMP-2在甲状腺肿瘤中的表达及其临床意义

目的:研究黏附分子CD44V6和基质金属蛋白酶-2(MMP-2)在甲状腺癌中的表达、相互关系及其与甲状腺癌侵袭转移的相关性.方法:采用SP免疫组化法检测114例甲状腺肿瘤组织中CD44V6和MMP-2的表达.结果:CD44V6和MMP-2在35例甲状腺乳头状癌(Papillary Thyroid Carcinoma,PTC)中阳性表达率分别为70.6%和73.5%,在41例甲状腺滤泡癌(Follicular Thyroid Carcinomas,FTC)中阳性表达率分别为70.7%和75.6%,均高于甲状腺腺瘤和结节性甲状腺肿组织中的表达,差异具有统计学意义(P＜0.05).在甲状腺癌组织中MMP-2和CD44V6的表达具有显著相关性(r=0.4828,P＜0.001).且两者表达与甲状腺癌的临床分期及有、无淋巴结转移显著相关.结论:CD44V6和MMP-2的表达与甲状腺癌分化程度、浸润和转移关系密切.CD44V6和MMP-2检测对甲状腺癌的诊断、分化程度、转移趋势和预后评估具有重要参考价值,是甲状腺癌侵袭、转移和预后判断的分子标志物.     

应用组织芯片技术研究Survivin基因蛋白在前列腺肿瘤组织中的表达及其意义

目的:应用组织芯片技术分析Survivin基因蛋白在人类前列腺癌组织、前列腺正常组织及前列腺良性痛变组织中的表达情况.方法:采用兔抗人survivin单克隆抗体的免疫组织化学ABC法,研究Survivin在不同前列腺组织的表达,并分析Survivin在不同前列腺组织中的表达差异.结果:免疫组化结果显示,前列腺癌组织与前列腺良性病变组织及正常前列腺组织中Survivin的表达相比呈显著性差异(P<0.05).结论:Survivin在前列腺癌组织中呈高表达,提示其可能对前列腺癌的发生或发展有重要作用.     

Survivin与VEGF在良、恶性胃溃疡组织中的表达及研究

目的:研究凋亡基因生存素(Survivin)及血管内皮生长因子(VEGF)在良、恶性胃溃疡中的表达及二者在溃疡型胃癌中的表达与临床病理特征之间的关系,分析二者在胃癌发生发展中的作用和在胃癌中表达的相关性。方法:应用免疫组化S-P染色检测Survivin及VEGF在良性胃溃疡,胃溃疡伴中-重度不典型增生和溃疡性胃癌中的表达,结合临床病理特征进行相关分析。结果:Survivin及VEGF在良性胃溃疡中的表达率分别为16.2%、24.3%,在胃溃疡伴中-重度不典型中的表达率分别为52.3%、45.5%,在溃疡型胃癌中的表达率分别为71.4%、55.4%,差异具有显著性(P0.01);Survivin和VEGF的表达与溃疡型胃癌的浸润深度、淋巴结转移、TNM分期具有相关性。Survivin和VEGF的表达亦呈正相关。结论:Survivin基因在溃疡型胃癌组织中的表达显著增高,是胃癌演变进程中的重要步骤,过表达Survivin可能提示预后不良。Survivin对胃癌的诊断及预后有潜在的应用价值。Survivin和VEGF在溃疡型胃癌的发生发展中起协同作用,动态随访二者对胃溃疡的演变可能有一定的价值,可作为判断肿瘤进程和浸润转移的生物学指标。Survivin及VEGF的联合检测可能对胃癌的综合治疗提供理论依据,对其进行深入研究有望为胃癌的诊疗开辟新的天地。     

Prognostic Significance of Nuclear Phospho-ATM Expression in Melanoma

UV radiation induced genomic instability is one of the leading causes for melanoma. Phosphorylation of Ataxia Telangiectasia Mutated (ATM) is one of the initial events that follow DNA damage. Phospho-ATM (p-ATM) plays a key role in the activation of DNA repair and several oncogenic pathways as well as in the maintenance of genomic integrity. The present study was therefore performed to understand the significance of p-ATM in melanoma progression and to correlate it with patient prognosis. Tissue microarray and immunohistochemical analysis were employed to study the expression of p-ATM in melanoma patients. A total of 366 melanoma patients (230 primary melanoma and 136 metastatic melanoma) were used for the study. Chi-square test, Kaplan-Meier, univariate and multivariate Cox regression analysis were used to elucidate the prognostic significance of p-ATM expression. Results revealed that both loss of, and gain in, p-ATM expression were associated with progression of melanoma from normal nevi to metastatic melanoma. Patients whose samples showed negative or strong p-ATM staining had significantly worse 5-year survival compared to patients who had weak to moderate expression. Loss of p-ATM expression was associated with relatively better 5-year survival, but the corresponding 10-year survival curve almost overlapped with that of strong p-ATM expression. p-ATM expression was found to be an independent prognostic factor for 5-year but not for 10-year patient survival. In conclusion our findings show that loss of p-ATM expression and gain-in p-ATM expression are indicators of worse melanoma patient survival.     

恶性卵巢肿瘤淋巴转移与相关基因表达

恶性卵巢肿瘤严重威胁着妇女健康和生命,其重要原因之一是易发生淋巴转移,给临床诊断和治疗带来困难。近年来国内外对恶性卵巢肿瘤淋巴转移的机制及其与相关基因表达进行了一系列的研究,现就这方面的进展作一综述。     

Annexin Ⅰ蛋白在人前列腺癌中的表达及临床意义

目的:探讨Annexin Ⅰ蛋白在前列腺癌组织中的表达及其与前列腺癌发生、发展、转移及预后的关系.方法:回顾性分析110例前列腺癌及40例前列腺增生组织中Annexin Ⅰ蛋白的表达与前列腺癌Gleason分级、年龄、临床分期、转移及预后的相关性.结果:前列腺癌患者的Armexin Ⅰ表达水平显著地低于良性前列腺增生症患者(P＜0.05);Annexin Ⅰ在低分化癌中表达较高,与高、中分化癌相比有显著性差异(P＜0.05);而高、中分化癌之间Annexin Ⅰ表达均较低,两者之间无显著性差异(P＞0.05).前列腺癌Annexin Ⅰ蛋白表达与年龄无关,而与临床分期、淋巴结转移及预后有关(P＜0.05).结论:Annexin Ⅰ蛋白表达下调与前列腺癌发生发展和预后密切相关,可作为反映前列腺癌生物学行为和判断预后的生物标记物.