Yi-Chien Lee

GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein

Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson’s disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3β because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3β. GSK-3β-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3β to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3β-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3β-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3β directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3β. Together, these data establish a novel upstream role for GSK-3β as one of several kinases associated with PTMs of key proteins known to be causal in PD.

VMY-1-103 is a novel CDK inhibitor that disrupts chromosome organization and delays metaphase progression in medulloblastoma cells

Medulloblastoma is the most prevalent of childhood brain malignancies, constituting 25% of childhood brain tumors. Craniospinal radiotherapy is a standard of care, followed by a 12-month regimen of multi-agent chemotherapy. For children less than 3 years of age, irradiation is avoided due to its destructive effects on the developing nervous system. Long-term prognosis is worst for these youngest children and more effective treatment strategies with a better therapeutic index are needed. VMY-1-103, a novel dansylated analog of purvalanol B, was previously shown to inhibit cell cycle progression and proliferation in prostate and breast cancer cells more effectively than purvalanol B. In the current study, we have identified new mechanisms of action by which VMY-1-103 affected cellular proliferation in medulloblastoma cells. VMY-1-103, but not purvalanol B, significantly decreased the proportion of cells in S phase and increased the proportion of cells in G2/M. VMY-1-103 increased the sub G1 fraction of apoptotic cells, induced PARP and caspase-3 cleavage and increased the levels of the Death Receptors DR4 and DR5, Bax and Bad while decreasing the number of viable cells, all supporting apoptosis as a mechanism of cell death. p21CIP1/WAF1 levels were greatly suppressed. Importantly, we found that while both VMY and flavopiridol inhibited intracellular CDK1 catalytic activity, VMY-1-103 was unique in its ability to severely disrupt the mitotic spindle apparatus significantly delaying metaphase and disrupting mitosis. Our data suggest that VMY-1-103 possesses unique antiproliferative capabilities and that this compound may form the basis of a new candidate drug to treat medulloblastoma.

Divergent brain changes in two audiogenic rat strains: A voxel-based morphometry and diffusion tensor imaging comparison of the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR)

Acoustically evoked seizures (e.g., audiogenic seizures or AGS) are common in models of inherited epilepsy and occur in a variety of species including rat, mouse, and hamster. Two models that have been particularly well studied are the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR) strains. Acute and repeated AGS, as well as comorbid conditions, displays a close phenotypic overlap in these models. Whether these similarities arise from convergent or divergent structural changes in the brain remains unknown. Here, we examined the brain structure of Sprague Dawley (SD) and Wistar (WIS) rats, and quantified changes in the GEPR-3 and WAR, respectively. Brains from adult, male rats of each strain (n=8-10 per group) were collected, fixed, and embedded in agar and imaged using a 7 tesla Bruker MRI. Post-acquisition analysis included voxel-based morphometry (VBM), diffusion tensor imaging (DTI), and manual volumetric tracing. In the VBM analysis, GEPR-3 displayed volumetric changes in brainstem structures known to be engaged by AGS (e.g., superior and inferior colliculus, periaqueductal grey) and in forebrain structures (e.g., striatum, septum, nucleus accumbens). WAR displayed volumetric changes in superior colliculus, and a broader set of limbic regions (e.g., hippocampus, amygdala/piriform cortex). The only area of significant overlap in the two strains was the midline cerebellum: both GEPR-3 and WAR showed decreased volume compared to their control strains. In the DTI analysis, GEPR-3 displayed decreased fractional anisotropy (FA) in the corpus callosum, posterior commissure and commissure of the inferior colliculus (IC). WAR displayed increased FA only in the commissure of IC. These data provide a biological basis for further comparative and mechanistic studies in the GEPR-3 and WAR models, as well as provide additional insight into commonalities in the pathways underlying AGS susceptibility and behavioral comorbidity.

Manganese-Enhanced MRI as a diagnostic and dispositional tool after mild-moderate blast TBI.

Traumatic brain injury (TBI) caused by explosive munitions, known as blast TBI, is the signature injury in recent military conflicts in Iraq and Afghanistan. Diagnostic evaluation of TBI, including blast TBI, is based on clinical history, symptoms, and neuropsychological testing, all of which can result in misdiagnosis or underdiagnosis of this condition, particularly in the case of TBI of mild-to-moderate severity. Prognosis is currently determined by TBI severity, recurrence, and type of pathology, and also may be influenced by promptness of clinical intervention when more effective treatments become available. An important task is prevention of repetitive TBI, particularly when the patient is still symptomatic. For these reasons, the establishment of quantitative biological markers can serve to improve diagnosis and preventative or therapeutic management. In this study, we used a shock-tube model of blast TBI to determine whether manganese-enhanced magnetic resonance imaging (MEMRI) can serve as a tool to accurately and quantitatively diagnose mild-to-moderate blast TBI. Mice were subjected to a 30 psig blast and administered a single dose of MnCl2 intraperitoneally. Longitudinal T1-magnetic resonance imaging (MRI) performed at 6, 24, 48, and 72 h and at 14 and 28 days revealed a marked signal enhancement in the brain of mice exposed to blast, compared with sham controls, at nearly all time-points. Interestingly, when mice were protected with a polycarbonate body shield during blast exposure, the marked increase in contrast was prevented. We conclude that manganese uptake can serve as a quantitative biomarker for TBI and that MEMRI is a minimally-invasive quantitative approach that can aid in the accurate diagnosis and management of blast TBI. In addition, the prevention of the increased uptake of manganese by body protection strongly suggests that the exposure of an individual to blast risk could benefit from the design of improved body armor.

Abstract B13: Perineural invasion in Ewing sarcoma—a novel mechanism and new therapeutic opportunities

Tumor dissemination and relapse are the major problems in Ewing sarcoma (ES) treatment, yet the mechanisms driving these processes are unknown. To elucidate the routes of ES metastatic spread, we used an orthotopic xenograft model. ES cells were injected into the gastrocnemius muscles of SCID/beige mice. Once the primary tumors reached the desired volume, they were excised by limb amputation. Subsequently, tumor dissemination was monitored by MRI and confirmed by histopathologic analysis. Interestingly, aside from typical hematogenous metastases, such as bone and lung lesions, we have also observed frequent perineural tumor dissemination manifested by the presence of migratory ES cells along the nerves adjacent to the primary tumors. This phenomenon was associated with formation of recurrent tumors at the amputation sites, as well as pelvic tumors with spine involvement. Interestingly, the level of perineural invasion (PNI) was dependent on the expression of neuropeptide Y (NPY) in ES cells. NPY is a neuronal protein released from peripheral sympathetic neurons, but also highly expressed in ES cells along with its receptors. The xenografts derived from ES cell lines not releasing endogenous NPY (TC71, TC32) exhibited frequent PNI in tumor-bearing limbs, as well as a high number of recurrent tumors at the surgery site and spine metastases (70% and 100% of mice with evidence of PNI for TC71 and TC32 xenografts, respectively). This phenomenon was less common in ES xenografts derived from NPY-rich SK-ES1 cells (17% of mice with signs of PNI). In line with these observations, NPY knockdown in SK-ES1 xenografts drastically accelerated formation of spinal tumors (60% of mice). Notably, in 40% of mice bearing SK-ES1/NPY shRNA xenografts the spinal tumors developed before the primary tumor growth was detectable at the site of ES cell injection. Thus, our in vivo experiments suggested that a lack of endogenous NPY in ES cells expressing high levels of its receptors triggers chemotactic effects of this peptide released from neighboring peripheral nerves, facilitating PNI. Indeed, in a transwell migration assay, NPY exerted significant chemotactic activity in SK-ES1/NPY shRNA cells, but not in the original SK-ES1 cell line. An even more profound chemotactic effect specific to the SK-ES1/NPY shRNA cells was observed with NPY-rich conditioned media obtained from neuroblastoma cells, which can serve as a model of peripheral sympathetic neurons. Further studies are required to determine which NPY receptors are responsible for its chemotactic properties. If the presence of perineural tumor growth is confirmed in human tumors, factors responsible for PNI in ES, e.g., NPY receptors, may become targets for novel therapies preventing disease dissemination and recurrence. Citation Format: Susana Galli, Sung-Hyeok Hong, Jason U. Tilan, Mina Adnani, Shiya Zhu, Yassi Fallah, Yi-Chien Lee, Olga Rodriguez, Chris Albanese, Ewa Izycka-Swieszewska, Joanna Kitlinska. Perineural invasion in Ewing sarcoma—a novel mechanism and new therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B13.

Abstract 1940: Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model

Neuroblastoma (NB) is a pediatric malignancy arising due to defects in sympathetic neuron differentiation. NB is a heterogeneous disease, with phenotypes ranging from spontaneously regressing to highly aggressive, incurable tumors. This clinical variability cannot be explained solely by genetic aberrations. Even in families with hereditary NB the penetrance of the disease is incomplete and the same genetic mutation often results in tumors with phenotypes varying from differentiating ganglioneuromas to undifferentiated, highly aggressive NBs. Thus, other, perhaps non-genetic factors can contribute to the disease development and modify its phenotype. Strikingly, the two factors promoting de-differentiation of NB cells and their malignant phenotype, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. Previously, using TH-MYCN mice as a model of aggressive NB, we have shown that an increase in maternal corticosterone levels during pregnancy attained by inserting slow release pellets resulted in increased tumor frequency in TH-MYCN offspring. The goal of the current study was to determine the effect of prenatal stress on NB metastasis. To this end, pregnant mice carrying TH-MYCN hemizygous offspring were subjected to chronic stress at embryonic days 10-17, the time of sympathetic neuroblast proliferation and differentiation. Two established stress paradigms were used - chronic unpredictable stress, in which mice were subjected daily to various stressors, and chronic cold stress comprising of daily 30 min exposure to cold. The phenotypes of the disease and its dissemination were compared between offspring of control and stressed mothers. The offspring from both prenatally stressed groups presented with more malignant disease, as manifested by the presence of advanced lung metastases disseminating from small primary tumors ( 1,000 mm3). Although not common, lung metastases occur preferentially in NB patients with MYCN amplification and are associated with significantly worse prognosis, as compared to patients with metastatic disease, but no pulmonary involvement (14 vs 43% 3-year event-free survival, respectively). Thus, the profound pulmonary dissemination observed in prenatally-stressed TH-MYCN mice mimics one of the most malignant NB phenotypes observed in human disease. Altogether, our data implicate maternal stress during pregnancy as a potential environmental factor modifying the effects of genetic aberrations and promoting malignant phenotype of NB. Citation Format: Sung Hyeok Hong, Larissa Wietlisbach, Susana Galli, Akanksha Mahajan, Shiya Zhu, Jason Tilan, Yichien Lee, Olga Rodriguez, Chris Albanese, Joanna Kitlinska. Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model [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 1940. doi:10.1158/1538-7445.AM2017-1940

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

Abstract 2478: Tumor hypoxia promotes Ewing sarcoma metastases in a mouse xenograft model

Ewing sarcoma (ES) is a pediatric tumor induced by EWS-ETS fusion proteins, most often EWS-FLI1. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear. Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of nonperfused areas within tumor tissue was associated with poor prognosis. In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes via changes in transcriptional activity of the EWS-FLI1 gene. However, despite this line of evidence, no direct proof for this hypoxia-induced ES progression and spread has been provided. Moreover, the mechanisms by which hypoxia could exert such effects are unknown. To fill this gap, we created an in vivo model of hypoxia in ES and tested its effect on tumor metastasis. SK-ES1 ES cells were injected into gastrocnemius muscles of SCID/beige mice. Once the tumors reached a volume of 250mm3, they were either excised (control) or subjected to femoral artery ligation (FAL) for 72h prior to excision, inducing ischemia of the lower hindlimb, thus creating tumor hypoxia. Then, the mice were monitored for metastases. The extent of the metastatic disease was assessed and compared between experimental groups based on periodic MRI, necropsy and histopathology findings. FAL resulted in profound tumor hypoxia, as evidenced by inhibition of primary tumor growth, severe tissue necrosis and positive staining for a hypoxyprobe, pimonidazole. However, despite the impaired growth of primary tumors, xenografts subjected to FAL were more metastatic. The involvement of hypoxic cells in metastases was evidenced by the accumulation of pimonidazole-positive cells (hypoxic at the time of FAL) in areas of tissue invasion and intravasation. Consequently, mice bearing FAL-treated tumors exhibited a decreased latency of metastases formation and an increase in their number from an average of 0.9 to 2.3 metastases per mouse in control and FAL groups, respectively. We also observed a change in the pattern of metastases, as FAL-treated tumors metastasized more often to distant organs (average of 0.3 organ metastases per mouse in control and 1.3 in FAL group). The hypoxia-induced metastases were most often observed in adrenal gland and spine (50% and 42% of mice in FAL group, respectively), while no such metastases were observed in the control group. Moreover, 100% of FAL-treated mice had signs of bone marrow invasion, while no tumor cells were detectable in bone marrow of control mice. This data provides the first-ever direct evidence for tumor hypoxia as a driver of ES metastases. Moreover, our model of tumor hypoxia in vivo provides an excellent opportunity to identify hypoxia-induced pathways involved in ES metastatic progression that subsequently may become novel therapeutic targets for this disease. Citation Format: Jason U. Tilan, Sung-Hyeok Hong, Susana Galli, Rachel Acree, Katherine Connors, Meredith Horton, Akanksha Mahajan, Larissa Wietlisbach, Yi-Chien Lee, Olga Rodriguez, Christopher Albanese, Joanna Kitlinska. Tumor hypoxia promotes Ewing sarcoma metastases in a mouse xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2478.

Abstract 3291: Prenatal stress increases neuroblastoma tumorigenesis in TH-MYCN mice model

Neuroblastomas (NB) are pediatric malignancies with heterogenous phenotypes, ranging from spontaneously regressing to highly aggressive, incurable tumors. Although NB is considered a genetic disease, its etiology and heterogeneity cannot be explained solely by genetic aberrations. NB arises due to defects in sympathetic neuron (SN) differentiation occurring during fetal development. Strikingly, the two factors promoting de-differentiation of NB cells, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. To test this hypothesis we used mice expressing MYCN oncogene under tyrosine hydroxylase promoter (TH-MYCN mice), which spontaneously develop NBs. To mimic stress, pregnant mothers carrying hemizygous TH-MYCN offspring were implanted with pellets containing either the main rodent glucocorticoid, corticosterone, or placebo at the time of neuroblast proliferation (embryonic days 10-20). Tumor frequency was compared between these two experimental groups and TH-MYCN offspring from intact pregnancies. Surprisingly, in pregnant mothers from the placebo group, physiological stress associated with experimental procedures alone (animal handling, blood collections, pellet insertion and anesthesia) was sufficient to elevate their corticosterone levels and increase tumorigenicity in their hemizygous TH-MYCN offspring from 32 to 64% (p = 0.03). A similar effect was observed in offspring of corticosterone-treated mothers with its levels comparable to mice eliciting a physiological stress response in the placebo group ( 1200ng/ml), indicating that only physiologically relevant levels of stress mediators can accurately recapitulate the stress response in animal models. Taken together, these findings support the role for prenatal stress in NB development, as well as implicate other pathologies associated with elevated levels of glucocorticoids in its etiology. Citation Format: Sung-Hyeok Hong, David Christian, Emily Trinh, Susana Galli, Meredith Horton, Yichien Lee, Christopher Albanese, Olga Rodriguez, Jason Ulip Tilan, Joanna Kitlinska. Prenatal stress increases neuroblastoma tumorigenesis in TH-MYCN mice model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3291. doi:10.1158/1538-7445.AM2015-3291