Matthew P. Wagoner

Metabolic Regulation of Neuronal Plasticity by the Energy Sensor AMPK

Long Term Potentiation (LTP) is a leading candidate mechanism for learning and memory and is also thought to play a role in the progression of seizures to intractable epilepsy. Maintenance of LTP requires RNA transcription, protein translation and signaling through the mammalian Target of Rapamycin (mTOR) pathway. In peripheral tissue, the energy sensor AMP-activated Protein Kinase (AMPK) negatively regulates the mTOR cascade upon glycolytic inhibition and cellular energy stress. We recently demonstrated that the glycolytic inhibitor 2-deoxy-D-glucose (2DG) alters plasticity to retard epileptogenesis in the kindling model of epilepsy. Reduced kindling progression was associated with increased recruitment of the nuclear metabolic sensor CtBP to NRSF at the BDNF promoter. Given that energy metabolism controls mTOR through AMPK in peripheral tissue and the role of mTOR in LTP in neurons, we asked whether energy metabolism and AMPK control LTP. Using a combination of biochemical approaches and field-recordings in mouse hippocampal slices, we show that the master regulator of energy homeostasis, AMPK couples energy metabolism to LTP expression. Administration of the glycolytic inhibitor 2-deoxy-D-glucose (2DG) or the mitochondrial toxin and anti-Type II Diabetes drug, metformin, or AMP mimetic AICAR results in activation of AMPK, repression of the mTOR pathway and prevents maintenance of Late-Phase LTP (L-LTP). Inhibition of AMPK by either compound-C or the ATP mimetic ara-A rescues the suppression of L-LTP by energy stress. We also show that enhanced LTP via AMPK inhibition requires mTOR signaling. These results directly link energy metabolism to plasticity in the mammalian brain and demonstrate that AMPK is a modulator of LTP. Our work opens up the possibility of using modulators of energy metabolism to control neuronal plasticity in diseases and conditions of aberrant plasticity such as epilepsy.

A Conspicuous Connection: Structure Defines Function for the Phosphatidylinositol-Phosphate Kinase Family

ABSTRACT The phosphatidylinositol phosphate (PIP) kinases are a unique family of enzymes that generate an assortment of lipid messengers, including the pivotal second messenger phosphatidylinositol 4,5-bisphosphate (PI4,5P2). While members of the PIP kinase family function by catalyzing a similar phosphorylation reaction, the specificity loop of each PIP kinase subfamily determines substrate preference and partially influences distinct subcellular targeting. Specific protein-protein interactions that are unique to particular isoforms or splice variants play a key role in targeting PIP kinases to appropriate subcellular compartments to facilitate the localized generation of PI4,5P2 proximal to effectors, a mechanism key for the function of PI4,5P2 as a second messenger. This review documents the discovery of the PIP kinases and their signaling products, and summarizes our current understanding of the mechanisms underlying the localized generation of PI4,5P2 by PIP kinases for the regulation of cellular events including actin cytoskeleton dynamics, vesicular trafficking, cell migration, and an assortment of nuclear events.

Type Iγ phosphatidylinositol phosphate kinase is required for EGF-stimulated directional cell migration

Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) modulates a plethora of cytoskeletal interactions that control the dynamics of actin assembly and, ultimately, cell migration. We show that the type Iγ phosphatidylinositol phosphate kinase 661 (PIPKIγ661), an enzyme that generates PI4,5P2, is required for growth factor but not G protein–coupled receptor–stimulated directional migration. By generating PI4,5P2 and regulating talin assembly, PIPKIγ661 modulates nascent adhesion formation at the leading edge to facilitate cell migration. The epidermal growth factor (EGF) receptor directly phosphorylates PIPKIγ661 at tyrosine 634, and this event is required for EGF-induced migration. This phosphorylation regulates the interaction between PIPKIγ661 and phospholipase Cγ1 (PLCγ1, an enzyme previously shown to be involved in the regulation of EGF-stimulated migration). Our results suggest that phosphorylation events regulating specific PIPKIγ661 interactions are required for growth factor–induced migration. These interactions in turn define the spatial and temporal generation of PI4,5P2 and derived messengers required for directional migration.

The Transcription Factor REST Is Lost in Aggressive Breast Cancer

The function of the tumor suppressor RE1 silencing transcription factor (REST) is lost in colon and small cell lung cancers and is known to induce anchorage-independent growth in human mammary epithelial cells. However, nothing is currently known about the role of this tumor suppressor in breast cancer. Here, we test the hypothesis that loss of REST function plays a role in breast cancer. To assay breast tumors for REST function, we developed a 24-gene signature composed of direct targets of the transcriptional repressor. Using the 24- gene signature, we identified a previously undefined RESTless breast tumor subtype. Using gene set enrichment analysis, we confirmed the aberrant expression of REST target genes in the REST–less tumors, including neuronal gene targets of REST that are normally not expressed outside the nervous system. Examination of REST mRNA identified a truncated splice variant of REST present in the REST–less tumor population, but not other tumors. Histological analysis of 182 outcome-associated breast tumor tissues also identified a subpopulation of tumors that lack full-length, functional REST and over-express the neuroendocrine marker and REST target gene Chromogranin A. Importantly, patients whose tumors were found to be REST–less using either the 24-gene signature or histology had significantly poorer prognosis and were more than twice as likely to undergo disease recurrence within the first 3 years after diagnosis. We show here that REST function is lost in breast cancer, at least in part via an alternative splicing mechanism. Patients with REST–less breast cancer undergo significantly more early disease recurrence than those with fully functional REST, regardless of estrogen receptor or HER2 status. Importantly, REST status may serve as a predictor of poor prognosis, helping to untangle the heterogeneity inherent in disease course and response to treatment. Additionally, the alternative splicing observed in REST–less breast cancer is an attractive therapeutic target.

A REST derived gene signature stratifies glioblastomas into chemotherapy resistant and responsive disease.

BackgroundGlioblastomas are the most common central nervous system neoplasia in adults, with 9,000 cases in the US annually. Glioblastoma multiformae, the most aggressive glioma subtype, has an 18% one-year survival rate, and 3% two year survival rate. Recent work has highlighted the role of the transcription factor RE1 Silencing Transcription Factor, REST in glioblastoma but how REST function correlates with disease outcome has not been described.MethodUsing a bioinformatic approach and mining of publicly available microarray datasets, we describe an aggressive subtype of gliomas defined by a gene signature derived from REST. Using this REST gene signature we predict that REST function is enhanced in advanced glioblastoma. We compare disease outcomes between tumors based on REST status and treatment regimen, and describe downstream targets of REST that may contribute to the decreased benefits observed with high dose chemotherapy in REM tumors.ResultsWe present human data showing that patients with “REST Enhanced Malignancies” (REM) tumors present with a shorter disease free survival compared to non-REM gliomas. Importantly, REM tumors are refractory to multiple rounds of chemotherapy and patients fail to respond to this line of treatment.ConclusionsThis report is the first to describe a REST gene signature that predicts response to multiple rounds of chemotherapy, the mainline therapy for this disease. The REST gene signature may have important clinical implications for the treatment of glioblastoma.

Induction of the RNA Regulator LIN28A Is Required for the Growth and Pathogenesis of RESTless Breast Tumors

The transcription factor RE1 silencing transcription factor (REST) is lost in approximately 20% of breast cancers. Although it is known that these RESTless tumors are highly aggressive and include all tumor subtypes, the underlying tumorigenic mechanisms remain unknown. In this study, we show that loss of REST results in upregulation of LIN28A, a known promoter of tumor development, in breast cancer cell lines and human breast tumors. We found that LIN28A was a direct transcriptional target of REST in cancer cells and that loss of REST resulted in increased LIN28A expression and enhanced tumor growth both in vitro and in vivo, effects that were dependent on heightened LIN28A expression. Tumors lacking REST expression were locally invasive, consistent with the increased lymph node involvement observed in human RESTless tumors. Clinically, human RESTless breast tumors also displayed significantly enhanced LIN28A expression when compared with non-RESTless tumors. Our findings therefore show a critical role for the REST-LIN28A axis in tumor aggression and suggest a causative relationship between REST loss and tumorigenicity in vivo.

Abstract LB-271: Loss of the transcription factor REST promotes breast cancer tumorigenicity in a LIN28-dependent manner

About 20% of the nearly 300,000 American women diagnosed with breast cancer this year will have a particularly aggressive form of the disease characterized by a lack of the transcription factor REST. These “RESTless” tumors are associated with particularly poor patient prognosis; 50% of patients with RESTless tumors experience disease recurrence within the first three years post-diagnosis, compared with less than 20% of patients whose tumors still express REST. Here, we use cell culture and xenograft models of RESTless breast cancer to demonstrate that REST loss causes the increased aggression of these tumors. Using lentiviral delivery of shRNAs, we generated several breast cell lines with a stable REST knockdown (“REST low ” cells; a non-targeting shRNA was used to generate control “REST norm ” cells). We find that REST knockdown increases focus formation in MCF10A, MCF7 and MDA-MB-231 cells. REST low MCF10A and MCF7 cells also show enhanced anchorage-independent growth in soft agar. We performed xenograft experiments with REST low and REST norm MCF7 cells; the REST low cells have a greatly enhanced capacity to form tumors in nude mice. We further explore the molecular mechanism underlying the tumorigenicity of cells lacking REST. We demonstrate that REST is a direct transcriptional repressor of the RNA binding protein LIN28 in breast cancer cell lines; as predicted, LIN28 expression is increased upon REST knockdown. Knockdown of LIN28 in REST low cells ablates the ability of REST low MCF7 cells to form foci in culture, colonies in soft agar, and tumors in nude mice. We therefore conclude that the aberrant upregulation of LIN28 that occurs when REST is lost is required for the tumorigenicity of REST low cells. Finally, we show that in human RESTless breast tumors, LIN28 expression is significantly increased. This is the first report to demonstrate that REST loss causes increased tumor aggression and to propose an underlying mechanism for the tumorigenicity of cells lacking REST. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-271. doi:1538-7445.AM2012-LB-271

Abstract 5020: Role of LIN28 in aggressive breast cancers lacking REST

Our lab has discovered a highly aggressive subset of breast cancers characterized by a lack of function of the tumor suppressor RE1 Silencing Transcription Factor (REST). REST is a transcriptional repressor controlling over 2000 neuronal genes, and has been identified as a tumor suppressor in lung and colon cancers. Our lab has discovered that REST function is lost in 20% of breast tumors and that its loss is associated with a significantly decreased disease-free survival and poor prognosis (Wagoner et al. poster, this meeting, CB4). In addressing the mechanisms underlying the aggressiveness of these RESTless tumors, we found that expression of the tumor promoter LIN28 is upregulated. We describe the direct regulation of LIN28 by REST in a range of mammary cell lines and show a correlation between REST loss and LIN28 overexpression in breast tumors. LIN28 inhibits maturation of the tumor suppressor microRNA let-7, which in turn suppresses expression of the oncogenes Myc and Ras. LIN28 overexpression has been found to promote metastasis in a mouse xenograft model of breast cancer. To examine the effects of REST loss and LIN28 expression on cellular transformation and motility, we have knocked down REST and LIN28 in a variety of breast cell lines (including T47D, MCF7, MDA-MB-231, 3T3 and NMuMG cells). Here, we describe the results of our colony formation, soft agar growth and motility assays. We recapitulate in vitro aspects of tumor aggression seen in the patient population, and we find that these processes are promoted by REST loss in a LIN28-dependent manner. We also describe xenograft models looking at the growth and metastasis of tumors that express or lack REST and LIN28. We conclude that REST loss promotes tumor aggressiveness at least in part via upregulation of its target gene LIN28. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5020.

Erratum: A conspicuous connection: Structure defines function for the phosphatidylinositol-phosphate kinase family (Critical Reviews in Biochemistry and Molecular Biology 42, (13-19))

Jessica N. Heck1∗, David L. Mellman1∗, Kun Ling1, Yue Sun, Matthew P. Wagoner1, Nicholas J. Schill2, and Richard A. Anderson1# A Conspicuous Connection: Structure Defines Function for the Phosphatidylinositol-Phosphate Kinase Family. 42: 15–19. ∗These authors contributed equally to this work and each should be considered first author. 1Program in Molecular and Cellular Pharmacology, University of Misconsin-Madison, Department of Pharmacology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706, USA 2Program in Cellular and Molecular Biology, University of WisconsinMadison, Department of Pharmacology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706, USA #Address correspondence to Richard Anderson. E-mail: raanders@wisc.edu