Myogenin modulates exercise endurance by altering skeletal muscle metabolism

The function of myogenic regulatory factors (MRFs) during adult life is not well understood. The requirement of one of these MRFs, myogenin (Myog), during embryonic muscle development suggests an equally important role in adult muscle. In this study, we have determined the function of myogenin during adult life using a conditional allele of Myog. In contrast to embryonic development, myogenin is not required for adult viability, and Myog-deleted mice exhibited no remarkable phenotypic changes during sedentary life. Remarkably, sedentary Myog-deleted mice demonstrated enhanced exercise endurance during involuntary treadmill running. Altered blood glucose and lactate levels in sedentary Myog-deleted mice after exhaustion suggest an enhanced glycolytic metabolism and an ability to excessively deplete muscle and liver glycogen stores. Traditional changes associated with enhanced exercise endurance, such as fiber type switching, and increased oxidative potential, were not detected in sedentary Myog-deleted mice. After long-term voluntary exercise, trained Myog-deleted mice demonstrated an enhanced adaptive response to exercise. Trained Myog-deleted mice exhibited superior exercise endurance associated with an increased proportion of slow-twitch fibers and increased oxidative capacity. In a parallel experiment, dystrophin-deficient young adult mice showed attenuated muscle fatigue following the deletion of Myog. These results demonstrate a novel and unexpected role for myogenin in modulating skeletal muscle metabolism.

CIP4 and Src in Promoting the Migration and Invasion of Breast Cancers

Cellular invasion represents a critical early step in the metastatic cascade, and many proteins have been identified as part of an “invasive signature.” The non-receptor tyrosine kinase Src is commonly upregulated in breast cancers, often in conjunction with overexpression of EGFR. Signaling from this pathway stimulates cell proliferation, migration, and invasion and frequently involves proteins that regulate the cytoskeleton. My data demonstrates that inhibition of Src, using the small-molecule inhibitor dasatinib, impairs cellular migration and invasion. Furthermore, Src inhibition sensitizes the cells to the effects of the chemotherapeutic doxorubicin resulting in dramatic, synergistic inhibition of proliferation with combination treatments. The Src-targeted protein CIP4 (Cdc42-interacting protein 4) associates with curved plasma membranes to scaffold complexes of Cdc42 and N-WASp. In these experiments, I show that CIP4 overexpression correlates with triple-negative biomarker status, cellular migration, and invasion of (breast cancer cells. Inhibition of CIP4 expression significantly decreases migration and invasion. Furthermore, I demonstrate the novel finding that CIP4 localizes to invadopodia, which are finger-like projections of the actin cytoskeleton that are associated with matrix degradation and cellular invasion. Depletion of CIP4 in invasive cells impairs the formation of invadopodia and the degradation of gelatin. Therefore, CIP4 is a critical component of the invasive phenotype acquired by human breast cancer cells. In this body of work, I propose a model in which CIP4 promotes actin polymerization by stabilizing the active conformation of N-WASp. CIP4 and N-WASp are both phosphorylated by Src, implicating this pathway in Src-dependent cytoskeletal rearragement. This represents a novel role for F-BAR proteins in migration and invasion.

THE CONSEQUENCES OF DISRUPTING THE MDM2-P53 BALANCE IN HEMATOPOIESIS

The bone marrow accommodates hematopoietic stem cells and progenitors. These cells provide an indispensible resource for replenishing the blood constituents throughout an organism’s life. A tissue with such a high turn-over rate mandates intact cycling checkpoint and apoptotic pathways to avoid inappropriate cell proliferation and ultimately the development of leukemias. p53, a major tumor suppressor, is a transcription factor that regulates cell cycle, and induces apoptosis and senescence. Mice inheriting a hypomorphic p53 allele in the absence of Mdm2, a p53 inhibitor, have elevated p53 cell cycle activity and die by postnatal day 13 due to hematopoietic failure. Hematopoiesis progresses normally during embryogenesis until it moves to the bone marrow in late development. Increased oxidative stress in the bone marrow compartment postnatally is the impediment for normal hematopoiesis via activation of p53. p53 in turn stimulates the generation of more reactive oxygen species and depletes bone marrow cellularity. Also, p53 exerts various defects on the hematopoietic niche by increasing mesenchymal lineage populations and their differentiation. Hematopoietic defects are rescued with antioxidants or when cells are cultured at low oxygen levels. Deletion of p16 partially rescues bone marrow cellularity and progenitors via a p53-independent pathway. Thus, although p53 is required to inhibit tumorigenesis, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis and survival during homeostasis.

THE ROLE OF TYROSINE PHOSPHORYLATION IN THE FUNCTIONS OF THE TUMOR SUPPRESSOR GPRC5A

The retinoic acid inducible G protein coupled receptor family C group 5 type A (GPRC5A) is expressed preferentially in normal lung tissue but its expression is suppressed in the majority of human non-small cell lung cancer cell lines and tissues. This differential expression has led to the idea that GPRC5A is a potential tumor suppressor. This notion was supported by the finding that mice with a deletion of the Gprc5a gene develop spontaneous lung tumors. However, there are various tumor cell lines and tissue samples, including lung, that exhibit higher GPRC5A expression than normal tissues and some reports by other groups that GPRC5A transfection increased cell growth and colony formation. Obviously, GPRC5A has failed to suppress the development of the tumors and the growth of the cell lines where its expression is not suppressed. Since no mutations were detected in the coding sequence of GPRC5A in 20 NSCLC cell lines, it’s possible that GPRC5A acts as a tumor suppressor in the context of some cells but not in others. Alternatively, we raised the hypothesis that the GPRC5A protein may be inactivated by posttranslational modification(s) such as phosphorylation. It is well established that Serine/Threonine phosphorylation of G protein coupled receptors leads to their desensitization and in a few cases Tyrosine phosphorylation of GPCRs has been linked to internalization. Others reported that GPRC5A can undergo tyrosine phosphorylation in the cytoplasmic domain after treatment of normal human mammary epithelial cells (HMECs) with epidermal growth factor (EGF) or Heregulin. This suggested that GPRC5A is a substrate of EGFR. Therefore, we hypothesized that tyrosine phosphorylation of GPRC5A by activation of EGFR signaling may lead to its inactivation. To test this hypothesis, we transfected human embryo kidney (HEK) 293 cells with GPRC5A and EGFR expression vectors and confirmed that GPRC5A can be tyrosine phosphorylated after activation of EGFR by EGF. Further, we found that EGFR and GPRC5A can interact either directly or through other proteins and that inhibition of the EGFR kinase activity decreased the phosphorylation of GPRA5A and the interaction between GPRC5A and EGFR. In c-terminal of GPRC5A, There are four tyrosine residues Y317, Y320, Y347, Y350. We prepared GPRC5A mutants in which all four tyrosine residues had been replaced by phenylalanine (mutant 4F) or each individual Tyr residue was replaced by Phe and found that Y317 is the major site for EGFR mediated phosphorylation in the HEK293T cell line. We also found that EGF can induce GPRC5A internalization both in H1792 transient and stable cell lines. EGF also partially inactivates the suppressive function of GPRC5A on cell invasion activity and anchorage-independent growth ability of H1792 stable cell lines. These finding support our hypothesis that GPRC5A may be inactivated by posttranslational modification- tyrosine phosphorylation.

Role and Regulation of EphA2 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets. The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found to be useful in 2 orthotopic models in vivo: 1) shRNA-pretreated Miapaca-2 cells, and 2) in vivo delivery of siRNA to established MPanc96 tumors. Silencing of EphA2 alone reduced tumor growth in Miapaca-2 cells. In MPanc96, only the combination treatment of gemcitabine plus siEphA2 significantly reduced tumor growth, as well as the number of lung and liver metastases. Taken together, these observations support EphA2 as a target for combination therapies for PDAC. The regulation of EphA2 was further explored with a focus on the role of Ras. K-Ras activating mutations are the most important initiating event in PDAC. We demonstrated that Ras regulates EphA2 expression through activation of MEK2 and phosphorylation of ERK. Downstream of ERK, silencing of the transcription factor AP-1 subunit c-Jun or inhibition of the ERK effector RSK caused a decrease in EphA2 expression, supporting their roles in this process. Further examination of Ras/MEK/ERK pathway modulators revealed that PEA-15, a protein that sequesters ERK to the cytoplasm, inhibited expression of EphA2. A significant inverse correlation between EphA2 and PEA-15 levels was observed in mouse models of PDAC. In cells where an EGFR inhibitor reduced phospho-Erk, expression of EphA2 was also reduced, indicating that changes in EphA2 levels may allow monitoring the effectiveness of anti-Ras/MEK/ERK therapies. In conclusion, EphA2 levels may be a good prognostic factor for anti-EGFR/anti-Ras therapies, and EphA2 itself is a relevant target for the development of new therapies.

COP 9 SIGNALOSOME SUBUNIT 6 STABILIZES COP1, A NOVEL E3 UBIQUITIN LIGASE FOR 14-3-3σ

14-3-3σ, a gene upregulated by p53 in response to DNA damage, exists as part of a positive-feedback loop which activates p53 and is a human cancer epithelial marker downregulated in various cancer types. 14-3-3σ levels are critical for maintaining p53 activity in response to DNA damage and regulating signal mediator such as Akt. Here, we identify Mammalian Constitutive Photomorphogenic 1 (COP1) as a novel E3 ubiquitin ligase for targeting 14-3-3σ through proteasome degradation. We show for the first time that COP9 signalosome subunit 6 (CSN6) associates with COP1 and is involved in 14-3-3σ ubiquitin-mediated degradation. Mechanistic studies show that CSN6 expression leads to stabilization of COP1 through reducing COP1 self-ubiquitination and decelerating COP1’s turnover rate. We also show that CSN6-mediated 14-3-3σ ubiquitination is compromised when COP1 is knocked down. Thus, CSN6 mediates 14-3-3σ ubiquitination through enhancing COP1 stability. Subsequently, we show that CSN6 causes 14-3-3σ downregulation, thereby activating Akt and promoting cell survival by suppressing FOXO, an Akt target, transcriptional activity. Also, CSN6 overexpression leads to increased cell growth, transformation and promotes tumorigenicity. Significantly, 14-3-3σ expression can correct the abnormalities mediated by CSN6 expression. These data suggest that the CSN6-COP1 axis is involved in 14-3-3σ degradation, and that deregulation of this axis will promote cell growth and tumorigenicity.

Altered Responses to Endoplasmic Reticulum Stress in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth most common cause of cancer-associated death in the United States. Little progress has been made in understanding how proteotoxic stress affects rapidly proliferating pancreatic tumor cells. Endoplasmic reticulum (ER) stress occurs when protein homeostasis in the ER lumen is perturbed. ER stress activates the unfolded protein response (UPR) to reduce the protein load in the ER. Under conditions of moderate ER stress, the UPR promotes cell cycle arrest which allows time for successful protein load reduction and enables cell survival. However, under conditions of high levels of ER stress the UPR induces cellular apoptosis. In this dissertation, I investigated the role of endoplasmic reticulum (ER) stress and its effects on the cell cycle in pancreatic cancer cells. Activation of the unfolded protein response after ER stress induction was determined by comparing expression of key UPR mediators in non-tumorigenic pancreatic ductal cells to pancreatic cancer cells. Two arms of the UPR were assessed: eIF2α/ATF4/CHOP and IRE1α/XBP1s. Pancreatic cancer cells exhibited altered UPR activation characterized by a delay in both phosphorylation of eIF2α and induction of spliced XBP1. Further evaluation of the UPR-mediated effects on cell cycle progression revealed that pancreatic cancer cells showed a compromised ability to inhibit G1 to S phase progression after ER stress. This reduced ability to arrest proliferation was found to be due to an impaired ability to downregulate cyclin D1, a key gatekeeper of the G1/S checkpoint. Abrogation of cyclin D1 repression was mediated through a slow induction of phosphorylation of eIF2α, a critical mediator of translational attenuation in response to ER stress. In conclusion, pancreatic cancer cells demonstrate a globally compromised ability to regulate the unfolded protein response. This deficiency results in reduced cyclin D1 repression through an eIF2α-mediated mechanism. These findings indicate that pancreatic cancer cells have increased tolerance for elevated ER stress compared to normal cells, and this tolerance results in continued tumor cell proliferation under proteotoxic conditions.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

Mechanisms regulating the p120-catenin/Kaiso pathway

The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.

A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

The Role of Cancer-Associated Fibroblasts in Lung Tumorigenesis

The extracellular milieu is rich in growth factors that drive tumor progression,but the mechanisms that govern tumor cell sensitivity to those ligands have notbeen fully defined. In this study, we address this question in mice that developmetastatic lung adenocarcinomas through the suppression of the microRNA-200 (miR-200) family. Cancer-associated fibroblasts (CAF) enhance tumorgrowth and invasion by secreting VEGF-A that binds to VEGFR1, a processrequired for tumor growth and metastasis in mice and correlated with a poorprognosis in lung adenocarcinoma patients. In this study, we discovered thatmiR-200 blocked CAF-induced tumor cell invasion by directly targetingVEGFR1 in tumor cells. In the context of previous studies, our findings suggestthat the miR-200 family is a point of convergence for diverse biologic processesthat regulate tumor cell proliferation, invasion, and metastasis; its target genesixdrive epithelial-to-mesenchymal transition (ZEB1 and ZEB2) and promotesensitivity to a potent tumor growth factor emanating from the microenvironment(VEGFR1). Clinical trials should focus not only on the role of VEGFR1 inangiogenesis but also on the expression and activation of VEGFR1 in tumorcells by stromal sources of VEGF-A in the tumor microenvironment as a targetfor metastasis prevention.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis Kim T. Cardenas The thymus and parathyroid glands originate from organ-specific domains of 3rd pharyngeal pouch (PP) endoderm. At embryonic day 11.5 (E11.5), the ventral thymus and dorsal parathyroid domains can be identified by Foxn1 and Gcm2 expression respectively. Neural crest cells, (NCCs) play a role in regulating patterning of 3rd PP endoderm. In addition, pharyngeal endoderm influences fate determination via secretion of Sonic hedgehog (Shh), a morphogen required for Gcm2 expression and generation of the parathyroid domain. Gcm2 is a downstream target of the transcription factor Tbx1, which in turn is positively regulated by Shh. Although initially expressed throughout pharyngeal pouch endoderm, Tbx1 expression is excluded from the thymus-specific domain of the 3rd PP by E10.5, but persists in the parathyroid domain. Based on these observations, we hypothesized that Tbx1 expression is non-permissive for thymus fate specification and that enforced expression of Tbx1 in the fetal thymus would impair thymus development. To test this hypothesis, we generated knock-in mice containing a Cre-inducible allele that allows for tissue-specific Tbx1 expression. Expression of the R26iTbx1 allele in fetal and adult thymus using Foxn1Cre resulted in severe thymus hypoplasia throughout ontogeny that persisted in the adult. Thymic epithelial cell (TEC) development was impaired as determined by immunohistochemical and FACS analysis of various differentiation markers. The relative level of Foxn1 expression in fetal TECs was significantly reduced. TECs in R26iTbx1/+ thymi assumed an almost universal expression of Plet-1, a marker associated with a TEC stem/progenitor cell fate. In addition, embryonic R26iTbx1/+ mice develop a perithymic mesechymal capsule that appears expanded compared to control littermates. Interestingly, thymi from neonatal and adult R26iTbx1/+ but not R26+/+ mice were encased in adipose tissue. This thymic phenotype also correlated with a decrease in thymocyte cellularity and aberrant thymocyte differentiation. The results to date support the conclusion that enforced expression of Tbx1 in TECs antagonizes their differentiation and prevents normal organogenesis via both direct and indirect effects.

CROSSTALK BETWEEN R1175 METHYLATION AND Y1173 PHOSPHORYLATION NEGATIVELY MODULATES EGFR-MEDIATED ERK ACTIVATION

Post-translational protein modifications are critical regulators of protein functions as they expand the signaling potentials of the modified proteins, leading to diverse physiological consequences. Currently, increasing evidence suggests that protein methylation is as important as other post-translational modifications in the regulation of various biological processes. This drives us to ask whether methylation is involved in the EGFR (epidermal growth factor receptor) signaling, a biological process extensively regulated by multiple post-translational modifications including phosphorylation, glycosylation and ubiquitination. We found that EGFR R1175 is methylated by a protein arginine methyltransferase named PRMT5. During EGFR activation, PRMT5-mediated R1175 methylation specifically enhances EGF-induced EGFR autophosphorylation at Y1173 residue. This novel modification crosstalk increases SHP1 recruitment to EGFR and suppresses EGFR-mediated ERK activation, resulting in inhibition of cell proliferation, migration, and invasion of EGFR-expressing cells. Based on these findings, we provide the first link between arginine methylation and tyrosine phosphorylation and identify R1175 methylation as an inhibitory modification specifically against EGFR-mediated ERK activation.

Mechanisms of Adenovirus-Mediated Autophagy

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered how viruses have evolved to manipulate autophagy for their benefit. Extensive studies of viral-induced autophagy provide a rationale to investigate other viruses, such as the adenovirus, which may be developed as part of a therapy against cancers resistant to apoptosis. Despite the present and relatively poor understanding of the mechanisms behind adenoviral-induced autophagy, adenovirus is a promising candidate, because of its ability to efficiently eradicate tumors. A better understanding of how the adenovirus induces autophagy will allow for the development of viruses with increased oncolytic potency. We hypothesized that adenovirus induces autophagy in order to aid in lysis. We found that replication, not infection, was required for adenovirus-mediated autophagy. Loss of function analysis of early genes revealed that, of the early genes tested, no single gene was sufficient to induce autophagy alone. Examination of cellular pathways for their role in autophagy during adenovirus infection revealed a function for the eIF2α pathway and more specifically the GCN2 kinase. Cells lacking GCN2 are more resistant to adenovirus-mediated autophagy in vitro; in vivo we also found these cells fail to undergo autophagy, but display more cell death. We believe that autophagy is a protective mechanism the cell employs during adenoviral infection, and in the in vivo environment, cells cannot recover from virus infection and are more susceptible to death. Congruently, infected cells deficient for autophagy through deletion of ATG5 are not able undergo productive cell lysis, providing evidence that the destruction of the cytoplasm and cell membrane through autophagy is crucial to the viral life cycle. This project is the first to describe a gene, other than a named autophagy gene, to be required for adenovirus- mediated autophagy. It is also the first to examine autophagic cell death as a means to aid in viral-induced cell lysis.