Phosphatase And Tensin Homolog On Chromosome Ten Research Articles

Multiple bronchial carcinoids associated with Cowden syndrome.

Cowden syndrome (CS) is a rare genetic condition due to the various germline mutations in the phosphatase and tensin homologue on chromosome ten (PTEN) tumour suppressor gene. As a result, CS is characterised by an increased risk of developing various benign and malignant tumours, such as thyroid, breast, endometrial and urogenital neoplasms, as well as gastrointestinal tract tumours. However, the neuroendocrine tumour association with CS is not elucidated yet. We present a case of a 46-year-old male patient diagnosed with testicular seminoma and follicular thyroid cancer in his medical history. Our patient met the clinical diagnostic criteria of Cowden syndrome. Genetic analysis established the clinical diagnosis; a known heterozygous PTEN mutation was detected [PTEN (LRG_311t1)c.388 C > T (p.Arg130Ter)]. Incidentally, he was also seen with multiple pulmonary lesions during his oncological follow-up. A video-assisted thoracoscopic left lingula wedge resection and later resections from the right lung were performed. Histological findings revealed typical pulmonary carcinoid tumours and smaller tumorlets. Somatostatin receptor SPECT-CT, 18F-FDG-PET-CT and 18F-FDOPA-PET-CT scans and endoscopy procedures could not identify any primary tumours in other locations. Our patient is the first published case of Cowden syndrome, associated with multifocal pulmonary carcinoids. Besides multiple endocrine neoplasia type 1, we propose Cowden syndrome as another hereditary condition predisposing to multiple pulmonary tumorlets and carcinoid tumours.

Effects of lentivirus-mediated RNA interference of HIF-1α and PTEN on oxygen-glucose deprivation injury in primary cultured rat neurons

To observe the effects of lentivirus-mediated RNA interference (RNAi) of hypoxia-inducible factor 1α (HIF-1α) and phosphatase and tensin homolog on chromosome ten (PTEN) on oxygen-glucose deprivation (OGD) injury in primary cultured rat neurons. Primary cultures of neonatal SD rat neurons were infected by lentiviral vectors carrying short hairpin RNA (shRNA) targeting HIF-1α or PTEN followed 4 days later by hypoxic exposure, and the control neurons were infected with the empty virus only with or without subsequent hypoxic exposure. Twenty-four hours after hypoxia, the interference efficiency was assessed with qRT-PCR, and lactate dehydrogenase (LDH) assay and AnnexinV-FITC/ PI assay were performed to detect neuronal damage and apoptosis. The expressions of the related proteins were determined with Western blotting. Lentivirus-mediated RNAi effectively silenced the mRNA expression of the target genes. HIF-1α silencing obviously aggravated the hypoxia-induced damage and apoptosis of the neurons, enhanced the expression of PTEN protein and significantly lowered the expressions of p-PTEN, p-AKT, NR2A and VEGFa (P < 0.05). PTEN silencing significantly alleviated hypoxia-induced damage and apoptosis of the neurons and increased the cellular expressions of p-PTEN and p-AKT (P < 0.05) without obviously affecting the expressions of HIF-1α, NR2A or VEGFa (P>0.05). An up-regulated expression of HIF-1α causes down-regulation of PTEN expression to protect primary cultured rat neurons against OGD injury.

PTEN alleviates maladaptive repair of renal tubular epithelial cells by restoring CHMP2A-mediated phagosome closure

Phosphatase and Tensin Homolog on chromosome Ten (PTEN) has emerged as a key protein that governs the response to kidney injury. Notably, renal adaptive repair is important for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. To test the role of PTEN in renal repair after acute injury, we constructed a mouse model that overexpresses PTEN in renal proximal tubular cells (RPTC) by crossing PTENfl-stop-fl mice with Ggt1-Cre mice. Mass spectrometry-based proteomics was performed after subjecting these mice to ischemia/reperfusion (I/R). We found that PTEN was downregulated in renal tubular cells in mice and cultured HK-2 cells subjected to renal maladaptive repair induced by I/R. Renal expression of PTEN negatively correlated with NGAL and fibrotic markers. RPTC-specific PTEN overexpression relieved I/R-induced maladaptive repair, as indicated by alleviative tubular cell damage, apoptosis, and subsequent renal fibrosis. Mass spectrometry analysis revealed that differentially expressed proteins in RPTC-specific PTEN overexpression mice subjected to I/R were significantly enriched in phagosome, PI3K/Akt, and HIF-1 signaling pathway and found significant upregulation of CHMP2A, an autophagy-related protein. PTEN deficiency downregulated CHMP2A and inhibited phagosome closure and autolysosome formation, which aggravated cell injury and apoptosis after I/R. PTEN overexpression had the opposite effect. Notably, the beneficial effect of PTEN overexpression on autophagy flux and cell damage was abolished when CHMP2A was silenced. Collectively, our study suggests that PTEN relieved renal maladaptive repair in terms of cell damage, apoptosis, and renal fibrosis by upregulating CHMP2A-mediated phagosome closure, suggesting that PTEN/CHMP2A may serve as a novel therapeutic target for the AKI to CKD transition.

Germline PTEN mutations are associated with a skewed peripheral immune repertoire in humans and mice.

Individuals with germline mutations in the gene encoding phosphatase and tensin homolog on chromosome ten (PTEN) are diagnosed with PTEN hamartoma tumor syndrome (PHTS) and are at high risk for developing breast, thyroid and other cancers and/or autoimmunity or neurodevelopmental issues including autism spectrum disorders. Although well recognized as a tumor suppressor, involvement of PTEN mutations in mediating such a diverse range of phenotypes indicates a more central involvement for PTEN in immunity than previously recognized. To address this, sequencing of the T-cell receptor variable-region β-chain was performed on peripheral blood from PHTS patients. Based on patient findings, we performed mechanistic studies in two Pten knock-in murine models, distinct from each other in cell compartment-specific predominance of Pten. We found that PTEN mutations in humans and mice are associated with a skewed T- and B-cell gene repertoire, characterized by increased prevalence of high-frequency clones. Immunological characterization showed that Pten mutants have increased B-cell proliferation and a proclivity towards increased T-cell reactivity upon Toll-like-receptor stimulation. Furthermore, decreases in nuclear but not cytoplasmic Pten levels associated with a reduction in expression of the autoimmune regulator (Aire), a critical mediator of central immune tolerance. Mechanistically, we show that nuclear PTEN most likely regulates Aire expression via its emerging role in splicing regulation. We conclude that germline disruption of PTEN, both in human and mouse, results in compromised central immune tolerance processes that may significantly impact individual stress responses and therefore predisposition to autoimmunity and cancer.

Overexpression of GATA4 enhances the antiapoptotic effect of exosomes secreted from cardiac colony-forming unit fibroblasts via miRNA221-mediated targeting of the PTEN/PI3K/AKT signaling pathway

BackgroundGATA4 is an early cardiac-specific transcription factor, and endogenous GATA4-positive cells play a critical role in cardioprotection after myocardial injury. As functional paracrine units of therapeutic cells, exosomes can partially reproduce the reparative properties of their parental cells. Here, we investigated the cardioprotective capabilities of exosomes derived from cardiac colony-forming unit fibroblasts (cCFU-Fs) overexpressing GATA4 (cCFU-FsGATA4) and the underlying mechanism through which these exosomes use microRNA (miRNA) delivery to regulate target proteins in myocardial infarction (MI).MethodsExosomes were harvested from cCFU-Fs by ultracentrifugation. miRNA arrays were performed to determine differential miRNA expression between exosomes derived from cCFU-FsGATA4 (GATA4-Exo) and control cCFU-Fs (NC-Exo). A dual-luciferase reporter assay confirmed that miR221 directly targets the 3′ untranslated region (UTR) of the phosphatase and tensin homolog on chromosome ten (PTEN) gene. Cardiac function and myocardial infarct size were evaluated by echocardiography and Masson trichrome staining, respectively.ResultsCompared with NC-Exo, GATA4-Exo increased the survival and reduced the apoptosis of H9c2 cells. Direct intramyocardial transplantation of GATA4-Exo at the border of the ischemic region following ligation of the left anterior descending (LAD) coronary artery significantly restored cardiac contractile function and reduced infarct size. Microarray analysis revealed significantly increased miR221 expression in GATA4-Exo. qPCR confirmed higher miR221 levels in H9c2 cells treated with GATA4-Exo than in those treated with NC-Exo. miR221 mimic-transfected H9c2 cells demonstrated a significantly higher survival rate following exposure to hypoxic conditions than those transfected with miR221 inhibitor. A dual-luciferase reporter gene assay confirmed the PTEN gene as a target of miR221. Western blot analysis showed that H9c2 cells treated with GATA4-Exo exhibited lower PTEN protein expression and higher p-Akt expression.ConclusionGATA4 overexpression enhances the protective effect of cCFU-F-derived exosomes on myocardial ischemic injury. In terms of the mechanism, it is at least partly due to the miR221 transferred by GATA4-Exo, which inhibits PTEN expression, activates the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway, and subsequently alleviates apoptosis of myocardial cells (CMs).

Selective Inhibition of the Immunoproteasome β5i Prevents PTEN Degradation and Attenuates Cardiac Hypertrophy.

Cardiac hypertrophy without appropriate treatment eventually progresses to heart failure. Our recent data demonstrated that the immunoproteasome subunit β5i promotes cardiac hypertrophy. However, whether β5i is a promising therapeutic target for treating hypertrophic remodeling remains unknown. Here, we investigated the effects of PR-957, a β5i-specific inhibitor, on angiotensin II (Ang II)–induced hypertrophic remodeling in the murine heart. The infusion of Ang II increased immunoproteasome chymotrypsin-like activity and β5i catalytic subunit expression in the heart, whereas PR-957 treatment fully blocked the enhanced immunoproteasome activity caused by Ang II. Moreover, the administration of PR-957 significantly suppressed Ang II–induced cardiac hypertrophy, fibrosis, and inflammation. Mechanistically, PR-957 treatment inhibited phosphatase and tensin homolog on chromosome ten (PTEN) degradation, thereby inhibiting multiple signals including AKT/mTOR, ERK1/2, transforming growth factor-β, and IKB/NF-kB. Furthermore, PTEN blocking by its specific inhibitor VO-OHpic markedly attenuated the inhibitory effect of PR-957 on Ang II–induced cardiac hypertrophy in mice. We conclude that PR-957 blocks PTEN degradation and activates its downstream mediators, thereby attenuating Ang II–induced cardiac hypertrophy. These findings highlight that PR-957 may be a potential therapeutic agent for Ang II-induced hypertrophic remodeling.

Coelonin, an Anti-Inflammation Active Component of Bletilla striata and Its Potential Mechanism.

Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism are still unclear. To uncover the active ingredients and potential molecular mechanism of the Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated and identified. It significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 μg/mL. The microarray data indicate that the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation level of cyclin dependent kinase inhibitor 1B (p27Kip1), another downstream molecule regulated by PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited NF-κB activation and p27Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor co-treatment analysis indicated that the inhibition of IL-1β, IL-6 and TNF-α expression by coelonin was independent of PTEN, whereas the inhibition of p27Kip1 degradation resulted in cell-cycle arrest in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo, which is one of the main active ingredients of Bletilla striata, deserves further study.

MicroRNA-92a antagonism attenuates lipopolysaccharide (LPS)-induced pulmonary inflammation and injury in mice through suppressing the PTEN/AKT/NF-κB signaling pathway

Overwhelming lung inflammation is a key feature of acute lung injury (ALI). MicroRNAs (miRNAs) have been implicated in the regulation diverse cellular processes including the inflammatory response. However, little is known about their functions and molecular involvement in regulating the inflammatory process in ALI. Herein, we established a lipopolysaccharide (LPS)-induced ALI mouse model and used miRNA microarray analysis to investigate and compare the miRNA expression profiles in mouse lung tissues. We found that miR-92a was markedly upregulated in the lung tissues of ALI mice compared with that in normal lung tissues. This upregulation of miR-92a in LPS-induced ALI mice was further confirmed in lung tissues, splenocytes and bronchoalveolar lavage fluid (BALF) by quantitative real-time PCR. Inhibition of miR-92a by injection with antagomir-92a markedly reduced LPS-induced pathological changes associated with lung inflammation, and reduces lung wet/dry ratio (W/D ratio), and Evans blue dye extravasation (an indicator of lung epithelial permeability). Moreover, inhibition of miR-92a ameliorated the inflammatory response by reducing the repression of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in lung tissues. In addition, we identified that miR-92a inhibited the phosphatase and tensin homolog on chromosome ten (PTEN) by binding to its 3ʹ-UTR in RAW264.7 murine macrophage cells. Western blot analysis demonstrated that inhibition of miR-92a may ameliorate inflammatory response through blocking PTEN/AKT/NF-κB signaling pathway in ALI mice. Collectively, these results have revealed a significant role of miR-92a in the lung inflammatory response associated with ALI in mice, and suggest that miR-92a may have potential as a prognostic indicator and novel therapeutic target for the treatment of ALI in future.

New Insights into Protein Kinase B/Akt Signaling: Role of Localized Akt Activation and Compartment-Specific Target Proteins for the Cellular Radiation Response.

Genetic alterations driving aberrant activation of the survival kinase Protein Kinase B (Akt) are observed with high frequency during malignant transformation and cancer progression. Oncogenic gene mutations coding for the upstream regulators or Akt, e.g., growth factor receptors, RAS and phosphatidylinositol-3-kinase (PI3K), or for one of the three Akt isoforms as well as loss of the tumor suppressor Phosphatase and Tensin Homolog on Chromosome Ten (PTEN) lead to constitutive activation of Akt. By activating Akt, these genetic alterations not only promote growth, proliferation and malignant behavior of cancer cells by phosphorylation of various downstream signaling molecules and signaling nodes but can also contribute to chemo- and radioresistance in many types of tumors. Here we review current knowledge on the mechanisms dictating Akt’s activation and target selection including the involvement of miRNAs and with focus on compartmentalization of the signaling network. Moreover, we discuss recent advances in the cross-talk with DNA damage response highlighting nuclear Akt target proteins with potential involvement in the regulation of DNA double strand break repair.

Abstract 601: Oncolytic and immune effects of RAPTOR: a novel oncolytic Herpes Simplex Virus 1 expressing PTEN-L for brain tumor therapy

Abstract Glioblastoma (GBM) is a World Health Organization grade IV astrocytoma with poor patient survival, characterized by extreme heterogeneity and invasiveness, and by resistance to radiation and chemotherapeutic drugs. Brain metastases are 2-3x more frequent than GBM, with breast cancer among the top 3 brain metastasizing tumor types. The tumor suppressor phosphatase and tensin homolog on chromosome ten (PTEN) is mutated or deleted in ~50% of all high grade gliomas and is lost in 33-49% of all breast cancers. We propose to target the dysregulated PTEN pathway in brain tumors to enhance patient survival. Oncolytic viruses are engineered to destroy tumor cells while sparing non-neoplastic cells, and can be designed to incorporate payloads that enhance their oncolytic effects. PTEN-L is a recently discovered, longer form of PTEN which is secreted and membrane permeable, and retains its phosphatase activity after secretion and re-entry into cells. We have generated a novel oncolytic herpesvirus (RAPTOR) that secretes PTEN-L from infected cells for the treatment of GBM and breast cancer brain metastases (BCBM). In vitro assays to evaluate virally mediated tumor cell killing, virus replication, transgene expression, and flow cytometry to evaluate intracellular pAkt and cell surface programmed death ligand 1 (PD-L1) were performed. In vivo studies using mice bearing orthotopic human GBM (nude mice) or murine BCBM (nude and FVB/N) were used to evaluate brain immune infiltrates and survival. Western blot analyses of infected cell lysates and conditioned media show that RAPTOR produces functional secreted PTEN-L, as confirmed by intracellular pAkt FACS. Tumor cell killing and replication were equivalent between RAPTOR and control HSVQ. In vivo, treatment of mice bearing established orthotopic BCBM revealed improved anti-tumor efficacy of RAPTOR. Significantly, 9/10 RAPTOR treated mice were long term survivors in immunocompetent FVB/N mice vs. 4/10 HSVQ treated mice (p=0.044). Treatment of nude mice bearing the same tumors did not produce long term survivors, implicating the development of antitumor immunity in RAPTOR treated animals. Flow cytometry revealed significant down regulation of the T-cell repressor PD-L1 on RAPTOR vs. HSVQ treated cells. Consistent with this, FACS of virus treated tumor bearing brain hemispheres revealed RAPTOR induced a significant influx in antigen presenting, NK, and CD8 T cells 7 days post treatment. Our findings demonstrate that RAPTOR enhances survival of brain tumor bearing mice using a two-pronged approach: lytic tumor cell death and immune cell education and activation against the brain tumor. RAPTOR inhibits virally induced PD-L1 expression on tumor cells, and could pave the way for a paradigm shift in immune therapy where immune checkpoint inhibition occurs locally within the tumor rather than globally, overriding toxicity of neutralizing antibodies. Citation Format: Luke Russell, Alexander Sprague, Alena Cristina Jaime-Ramirez, Chelsea Bolyard, Yeshavanth Banasavadi-Siddegowda, JiYoung Yoo, Pin Yi Wang, Timothy Cripe, Balveen Kaur. Oncolytic and immune effects of RAPTOR: a novel oncolytic Herpes Simplex Virus 1 expressing PTEN-L for brain tumor therapy [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 601. doi:10.1158/1538-7445.AM2017-601

STAT1 Inhibits MiR‐181a Expression to Suppress Colorectal Cancer Cell Proliferation Through PTEN/Akt

Signal transducers and activators of transcription 1 (STAT1) exhibits tumor-suppressor properties by inhibiting oncogenic pathways and promoting tumor immunosurveillance. MicroRNAs, a group of non-coding endogenous ones, may regulate gene expression and plays specific roles in tumorigenesis. Recently, miR-181a has been reported to be associated with poor prognosis of colorectal cancer (CRC). Using human colorectal cancer cell lines, we demonstrated that STAT1 suppresses both LoVo and SW480 cell growth by down-regulating miR-181a. STAT1 regulates the expression of miR-181a through binding to the elements in the miR-181a's promoter region. Further, we revealed that miR-181a accelerates CRC cell proliferation through phosphatase and tensin homolog on chromosome ten (PTEN). In addition, PTEN protein was upregulated in response to STAT1 overexpression or miR-181a inhibition, downregulated in response to STAT1 knockdown or miR-181a overexpression. Without changes on the AKT protein level, p-AKT was downregulated by STAT1 overexpression or miR-181a inhibition while upregulated by STAT1 knockdown or miR-181a overexpression, indicating PTEN/Akt pathway activated in STAT1/miR-181a regulation of CRC cell proliferation. Taken together, our findings shed new light on the STAT1/miR-181a/PTEN pathway in colorectal cancer and add new insight regarding the carcinogenesis of colorectal cancer. J. Cell. Biochem. 118: 3435-3443, 2017. © 2017 Wiley Periodicals, Inc.

Long-term consequences of conditional genetic deletion of PTEN in the sensorimotor cortex of neonatal mice

Targeted deletion of the phosphatase and tensin homolog on chromosome ten (PTEN) gene in the sensorimotor cortex of neonatal mice enables robust regeneration of corticospinal tract (CST) axons following spinal cord injury as adults. Here, we assess the consequences of long-term conditional genetic PTEN deletion on cortical structure and neuronal morphology and screen for neuropathology. Mice with a LoxP-flanked exon 5 of the PTEN gene (PTENf/f mice) received AAV-Cre injections into the sensorimotor cortex at postnatal day 1 (P1) and were allowed to survive for up to 18months. As adults, mice were assessed for exploratory activity (open field), and motor coordination using the Rotarod®. Some mice received injections of Fluorogold into the spinal cord to retrogradely label the cells of origin of the CST. Brains were prepared for neurohistology and immunostained for PTEN and phospho-S6, which is a downstream marker of mammalian target of rapamycin (mTOR) activation. Immunostaining revealed a focal area of PTEN deletion affecting neurons in all cortical layers, although in some cases PTEN expression was maintained in many small–medium sized neurons in layers III–IV. Neurons lacking PTEN were robustly stained for pS6. Cortical thickness was significantly increased and cortical lamination was disrupted in the area of PTEN deletion. PTEN-negative layer V neurons that give rise to the CST, identified by retrograde labeling, were larger than neurons with maintained PTEN expression, and the relative area occupied by neuropil vs. cell bodies was increased. There was no evidence of tumor formation or other neuropathology. Mice with PTEN deletion exhibited open field activity comparable to controls and there was a trend for impaired Rotarod performance (not statistically significant). Our findings indicate that early postnatal genetic deletion of PTEN that is sufficient to enable axon regeneration by adult neurons causes neuronal hypertrophy but no other detectable neuropathology.

AIF inhibits tumor metastasis by protecting PTEN from oxidation.

Apoptosis-inducing factor (AIF) exerts dual roles on cell death and survival, but its substrates as a putative oxidoreductase and roles in tumorigenesis remain elusive. Here, we report that AIF physically interacts with and inhibits the oxidation of phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor susceptible for oxidation-mediated inactivation. More intriguingly, we also identify PTEN as a mitochondrial protein and the ectopic expression of mitochondrial targeting sequence-carrying PTEN almost completely inhibits Akt phosphorylation in PTEN-deficient cells. AIF knockdown causes oxidation-mediated inactivation of the lipid phosphatase activity of PTEN, with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK-3β, and activation of β-catenin signaling in cancer cells. Through its effect on β-catenin signaling, AIF inhibits epithelial–mesenchymal transition (EMT) and metastasis of cancer cells in vitro and in orthotopically implanted xenografts. Accordingly, the expression of AIF is correlated with the survival of human patients with cancers of multiple origins. These results identify PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis.

Abstract 4943: Visualization of endogenous melanoma initiation and progression using intravital microscopy

Abstract The death rates for malignant metastatic melanoma continue to rise because melanoma is largely refractory to existing therapies. Loss or inactivation of the tumor suppressor PTEN (Phosphatase and Tensin Homolog on Chromosome Ten) is observed in 40-50% of melanoma, and BRAF and PTEN mutations exist coincidentally in approximately 20% of melanoma cases. In mouse GEMM models, these mutations cooperate to promote metastatic melanomagenesis. However, existing methods to activate these lesions with Tyr-CreERT2 and topical tamoxifen application yields multifocal primary tumors whose origins cannot be accurately determined. To improve this model, we combined a tdTomatoLSL allele that serves as a visual marker of Cre recombination with Tyr-CreERT2;BrafCA;Ptenlox/lox to produce an animal where we can reproducibly initiate tumorigenesis in a spatiotemporally controllable manner by transiently applying extremely low doses (&amp;lt;20 nM) of tamoxifen to the dermis of the ear. Due to melanocyte-specific expression of the fluorescent protein tdTomato, we can track tumor progression from endogenous cells using two-photon intravital imaging. Starting from a single primary tumor on the ear, we can detect distant metastases in cervical lymph nodes and lungs via macroscopic fluorescent imaging, qRT-PCR and histology. We are using this model in combination with targeted therapy to investigate the effects of selective molecular inhibitors on the growth and metastasis of endogenous BRAFV600E/PTEN-/- metastatic melanoma at the cellular level. Preliminary studies using intravital imaging and a selective MEK1/2 inhibitor, GSK212 (Trametinib, FDA approved treatment for metastatic melanoma) show that targeted therapy causes heterogeneous reorganization of the tumor cells as early as 3 days post-treatment. After several weeks of continuous treatment, the remaining, resistant tumor cells are highly correlated spatially with bundled collagen structures detected by second harmonic generation signal, suggesting that cellular milieu strongly influences drug response. In addition, GSK212 treated animals still had lung macro-metastatic tumors, indicating that despite continuous MEK1/2 inhibition, metastatic tumor growth in the lung was still possible. In summary, we have developed an inducible endogenous model of melanoma metastasis that can be used to directly investigate both heterogeneous effects of targeted therapy at the cellular level and the efficacy of targeted therapy against metastasis. Citation Format: James E. Bear, Hailey E. Brighton, Norman E. Sharpless, David B. Darr, Kelly S. Clark. Visualization of endogenous melanoma initiation and progression using intravital microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2014-4943

Megalencephaly and hemimegalencephaly: breakthroughs in molecular etiology.

Megalencephaly (MEG) is a developmental disorder characterized by brain overgrowth that occurs due to either increased number or size of neurons and glial cells. The former may be due to either increased neuronal proliferation or decreased apoptosis. The degree of brain overgrowth may be extensive, ranging from generalized MEG affecting the entire cortex-as with mutations in PTEN (phosphatase and tensin homolog on chromosome ten)-to unilateral hemispheric malformations-as in classic hemimegalencephaly (HME). On the other hand, some lesions are more focal or segmental. These developmental brain abnormalities may occur in isolation in some individuals, whereas others occur in the context of a syndrome involving dysmorphic features, skin findings, or other organ system involvement. Brain overgrowth disorders are often associated with malformations of cortical development, resulting in increased risk of epilepsy, intellectual disability, and autistic features, and some are associated with hydrocephalus. The past few years have witnessed a dramatic leap in our understanding of the molecular basis of brain overgrowth, particularly the identification of mosaic (or post-zygotic) mutations in core components of key cellular pathways such as the phosphatidylinositol 3-kinase (PI3K)-vakt murine thymoma viral oncogene homolog (AKT)-mTOR pathway. These molecular insights have broadened our view of brain overgrowth disorders that now appear to span a wide spectrum of overlapping phenotypic, neuroimaging, and neuropathologic features and molecular pathogenesis. These molecular advances also bring to light the possibility of pathway-based therapies for these often medically devastating developmental disorders.

Altered Expression of PTEN and Its Major Regulator MicroRNA-21 in Pulmonary Neuroendocrine Tumors

BackgroundPhosphatase and tensin homolog on chromosome ten (PTEN) is one of the most frequently inactivated tumor suppressors in various tumor types. MicroRNA-21 (miR-21) may affect tumor progression by post-transcriptional repression of expression of tumor suppressors, such as PTEN. This study was conducted to evaluate the significance of PTEN expression in pulmonary neuroendocrine (NE) tumors and to analyze the relationship between PTEN and miR-21 expressions.MethodsExpressions of PTEN and miR-21 were investigated by immunohistochemistry and real time reverse transcription-polymerase chain reaction, respectively, in 75 resected pulmonary NE tumors (23 typical carcinoids [TCs], nine atypical carcinoids [ACs], 22 large cell NE carcinomas [LCNECs], and 21 small cell lung carcinomas [SCLCs]).ResultsLoss of PTEN expression was observed in four of 23 TCs (17.4%), four of nine ACs (44.4%), 16 of 22 LCNECs (72.7%) and nine of 21 SCLCs (42.9%) (p=.025). The expression level of miR-21 was significantly higher in high-grade NE carcinomas than in carcinoid tumors (p<.001). PTEN expression was inversely correlated with miR-21 expression (p<.001).ConclusionsThis study suggests that aberrant expression of PTEN in relation to miR-21 may represent an important step in the development and progression of pulmonary NE tumors.

Colonic manifestations ofPTENhamartoma tumor syndrome: Case series and systematic review

To investigate our clinical experience with the colonic manifestations of phosphatase and tensin homolog on chromosome ten (PTEN) hamartoma tumor syndrome (PHTS) and to perform a systematic literature review regarding the same. This study was approved by the appropriate institutional review board prior to initiation. A clinical genetics database was searched for patients with PHTS or a component syndrome that received gastrointestinal endoscopy or pathology interpretation at our center. These patient's records were retrospectively reviewed for clinical characteristics (including family history and genetic testing), endoscopy results and pathology findings. We also performed a systematic review of the literature for case series of PHTS or component syndromes that reported gastrointestinal manifestations and investigations published after consensus diagnostic criteria were established in 1996. These results were compiled and reported. Eight patients from our institution met initial inclusion criteria. Of these, 5 patients underwent 4.2 colonoscopies at mean age 45.8 ± 10.8 years. All were found to have colon polyps during their clinical course and polyp histology included adenoma, hyperplastic, ganglioneuroma and juvenile. No malignant lesions were identified. Two had multiple histologic types. One patient underwent colectomy due to innumerable polyps and concern for future malignant potential. Systematic literature review of PHTS patients undergoing endoscopy revealed 107 patients receiving colonoscopy at mean age 37.4 years. Colon polyps were noted in 92.5% and multiple colon polyp histologies were reported in 53.6%. Common polyp histologies included hyperplastic (43.6%), adenoma (40.4%), hamartoma (38.3%), ganglioneuroma (33%) and inflammatory (24.5%) polyps. Twelve (11.2%) patients had colorectal cancer at mean age 46.7 years (range 35-62). Clinical outcomes secondary to colon polyposis and malignancy were not commonly reported. PHTS has a high prevalence of colon polyposis with multiple histologic types. It should be considered a mixed polyposis syndrome. Systematic review found an increased prevalence of colorectal cancer and we recommend initiating colonoscopy for colorectal cancer surveillance at age 35 years.

Modulation of microRNAs in hypertension-induced arterial remodeling through the β1 and β3-adrenoreceptor pathways

BackgroundDysregulation of microRNAs (miRNAs) in arterial dysfunction and hypertension has not been extensively investigated yet. This project determined the effects of two anti-hypertensive β1 adrenergic selective blockers on miRNA expression in the Dahl Salt Sensitive (DSS) hypertensive rat model. Methods and resultsMicroarray analysis showed that a set of miRNAs is differently expressed in the aorta of high salt (HS) treated rats with miR-320 increased and miR-26b and -21 decreased. All of these changes were reverted to normal by nebivolol (NEB, a β1 selective-blocker and β3 activator). The selective β3-adrenoceptor antagonist S-(−)-cyanopindolol (Syc) counteracted the effect of NEB on these miRNAs. Atenolol (ATN, a pure β1-blocker) combined with specific β3 agonist BRL37344 restored the expression of all three miRNAs, similar to NEB, while ATN alone had only a partial effect on miR-320 expression. Computational analysis found Insulin Growth Factor-1 Receptor (IGF1R) as a putative target of miR-320, and Phosphatase and tensin homolog on chromosome ten (PTEN) as a putative target of miR-26b and -21. The targets were verified by luciferase reporter assays. Inhibition of miR-320 by an antisense inhibitor or NEB increased IGF1R expression, while miR-320 overexpression reversed the effect of NEB. Overexpression of miR-26b or -21 or NEB decreased PTEN levels, while inhibition of miR-26b or -21 attenuated the effect of NEB. HS diet induced downregulation of IGF1R and upregulation of PTEN in the aorta. NEB normalized the aberrant expression of IGF1R and PTEN and also improved the impairment of vascular AKT/eNOS signaling. Moreover, both NEB and ATN showed to have protective effects on salt-induced hypertension, oxidative stress, and vascular remodeling. NEB had a greater effect than ATN. ConclusionsOur data supports a differential miRNA expression profile in salt-induced hypertension. Manipulation of dysregulated miRNAs by β-blockers may substantially induce alterations of gene expression and prevent arterial dysfunction and remodeling.

Overexpression of calcium-activated potassium channels underlies cortical dysfunction in a model of PTEN -associated autism

De novo phosphatase and tensin homolog on chromosome ten (PTEN) mutations are a cause of sporadic autism. How single-copy loss of PTEN alters neural function is not understood. Here we report that Pten haploinsufficiency increases the expression of small-conductance calcium-activated potassium channels. The resultant augmentation of this conductance increases the amplitude of the afterspike hyperpolarization, causing a decrease in intrinsic excitability. In vivo, this change in intrinsic excitability reduces evoked firing rates of cortical pyramidal neurons but does not alter receptive field tuning. The decreased in vivo firing rate is not associated with deficits in the dendritic integration of synaptic input or with changes in dendritic complexity. These findings identify calcium-activated potassium channelopathy as a cause of cortical dysfunction in the PTEN model of autism and provide potential molecular therapeutic targets.

Phosphodiesterase III Inhibition Increases cAMP Levels and Augments the Infarct Size Limiting Effect of a DPP-4 Inhibitor in Mice with Type-2 Diabetes Mellitus

We assessed whether phosphodiesterase-III inhibition with cilostazol (Cil) augments the infarct size (IS)-limiting effects of MK0626 (MK), a dipeptidyl-peptidase-4 (DPP4) inhibitor, by increasing intracellular cAMP in mice with type-2 diabetes. Db/Db mice received 3-day MK (0, 1, 2 or 3 mg/kg/d) with or without Cil (15 mg/kg/d) by oral gavage and were subjected to 30 min coronary artery occlusion and 24 h reperfusion. Cil and MK at 2 and 3 mg/kg/d significantly reduced IS. Cil and MK had additive effects at all three MK doses. IS was the smallest in the MK-3+Cil. MK in a dose dependent manner and Cil increased cAMP levels (p < 0.001). cAMP levels were higher in the combination groups at all MK doses. MK-2 and Cil increased PKA activity when given alone; however, PKA activity was significantly higher in the MK-2+Cil group than in the other groups. Both MK-2 and Cil increased myocardial levels of Ser(133) P-CREB, Ser(523) P-5-lipoxygenase, Ser(473)P-Akt and Ser(633) P-eNOS. These levels were significantly higher in the MK-2+Cil group. Myocardial PTEN (Phosphatase and tensin homolog on chromosome ten) levels were significantly higher in the Db/Db mice compared to nondiabetic mice. MK-2 and Cil normalized PTEN levels. PTEN levels tended to be lower in the combination group than in the MK and Cil alone groups. MK and Cil have additive IS-limiting effects in diabetic mice. The additive effects are associated with an increase in myocardial cAMP levels and PKA activity with downstream phosphorylation of Akt, eNOS, 5-lipoxygenase and CREB and downregulation of PTEN expression.