Barry Schoenike

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.

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.

Quantitative sense-specific determination of murine coronavirus RNA by reverse transcription polymerase chain reaction.

Abstract In many applications, it is useful to know the sense and amount of viral RNAs present in a sample. In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. However, in practice, it has been shown that such assays are prone to artifacts, such as non-specific priming, which drastically diminish their reliability. Using murine coronavirus MHV-4 as a model, we describe and validate several modifications of the RT-PCR procedure which eliminate these artifacts. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. The assays described may be used to determine the sense and abundance of any viral or host RNA of interest in complex biological specimens.

Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

ABSTRACT Loss of repressor element 1 silencing transcription factor (REST) occurs in 20% of breast cancers and correlates with a poor patient prognosis. However, the molecular basis for enhanced malignancy in tumors lacking REST (RESTless) is only partially understood. We used multiplatform array data from the Cancer Genome Atlas to identify consistent changes in key signaling pathways. Of the proteins screened in the reverse-phase protein array, we found that insulin receptor substrate 1 (IRS1) is the most highly upregulated protein in RESTless breast tumors. Analysis of breast tumor cell lines showed that REST directly represses IRS1, and cells lacking REST have increased levels of IRS1 mRNA and protein. We find that the upregulation of IRS1 function is both necessary and sufficient for enhanced signaling and growth in breast cancer cells lacking REST. IRS1 overexpression is sufficient to phenocopy the enhanced activation of the signaling hubs AKT and mitogen-activated protein kinase (MAPK) of MCF7 cells lacking REST. Loss of REST renders MCF7 and MDA-MB-231 breast tumor cells dependent on IRS1 activity for colony formation in soft agar. Inhibition of the type 1 insulin-like growth factor receptor (IGF1R) reduces the enhanced signaling, growth, and migration in breast tumor cells that occur upon REST loss. We show that loss of REST induces a pathogenic program that works through the IGF1R/IRS1 pathway.

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of Apc(Min) (/+) ROSA11 Gene Trap Mice.

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in ApcMin/+ mice. The insertion site of the β-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors.

Altered circadian rhythms and oscillation of clock genes and sirtuin 1 in a model of sudden unexpected death in epilepsy

Circadian rhythms are affected in many neurological disorders. Although sleep disturbances are known in epilepsy, data on circadian rhythm disturbances in epilepsy are sparse. Here, we examined diurnal and circadian rest‐activity and sleep‐wake patterns in Kcna1‐null mice, which exhibit spontaneous recurrent seizures and are a model of sudden unexpected death in epilepsy. Furthermore, we sought to determine whether seizures or aberrant oscillation of core clock genes and a regulator, sirtuin 1 (Sirt1), is associated with disrupted rhythms.

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.