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

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

Enhanced immunoprecipitation techniques for the identification of RNA-binding protein partners: IGF2BP1 interactions in mammary epithelial cells

RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested exclusion-based purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro

A Phenotypic Mouse Model of Basaloid Breast Tumors

Chemotherapeutic strategies that target basal-like breast tumors are difficult to design without understanding their cellular and molecular basis. Here, we induce tumors in mice by carcinogen administration, creating a phenocopy of tumors with the diagnostic and functional aspects of human triple negative disease (including EGFR expression/lack of erbB, estrogen-independent growth and co-clustering of the transcriptome with other basaloid models). These tumor strains are a complement to established mouse models that are based on mutations in Brca1 and/or p53. Tumors comprise two distinct cell subpopulations, basal and luminal epithelial cells. These cell fractions were purified by flow cytometry, and only basal cell fractions found to have tumor initiating activity (cancer stem cells). The phenotype of serially regenerated tumors was stable, and irrespective of tumor precursor cell. Tumors were passaged entirely in vivo and serial generations tested for their phenotypic stability. The relative chemo-sensitivity of basal and luminal cells were evaluated. Upon treatment with anthracycline, tumors were effectively de-bulked, but recurred; this correlated with maintenance of a high rate of basal cell division throughout the treatment period. Thus, these tumors grow as robust cell mixtures of basal bipotential tumor initiating cells alongside a luminal majority, and the cellular response to drug administration is dominated by the distinct biology of the two cell types. Given the ability to separate basal and luminal cells, and the discovery potential of this approach, we propose that this mouse model could be a convenient one for preclinical studies

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

<p>Abstract</p> <p>Background</p> <p>Glioblastomas 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.</p> <p>Method</p> <p>Using 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.</p> <p>Results</p> <p>We 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.</p> <p>Conclusions</p> <p>This 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.</p

KRT5+ DTumors parallel human basal-like breast tumors by functional and molecular assay.

<p>(A) <i>Mixed lineage tumors</i>. 19 primary tumors were double stained with lineage-specific markers (keratin 5, basal cells, stained red; keratin 8, luminal cells, stained green) and 17 primary tumors (90%) comprise ≥10% K5-positive cells, as well as K8-positive cells, indicating that these tumors are bi-lineal. Two representative images from two different primary tumors are shown. Scale bar = 50 µm. (B) <i>Estrogen-independence.</i> The appearance of palpable tumor masses was measured after transplanting 10,000 tumor cells into either control or ovarectomized three-week old BALB/c recipients (representative of 3 strains of primary tumor). Re-growth of tumors is no different in recipients grafted at 3 weeks of age. Paraffin sections from tumors removed from ovariectomized hosts were compared to tumors from normal hosts, by double staining for K5 (green) and K8 (red). There was no difference in their basal/luminal constituent cell types. (C) <i>Experimental tumors are ERα/PR-negative</i>. Paraffin sections from normal virgin BALB/c mammary glands (MG), together with an example of a primary tumor (Primary T) and a secondary tumor (Second T), were stained for ERα and PR, and counterstained as indicated with either K5 (basal) or a DAPI nuclear stain. (The specificity of the anti-PR-A staining procedure is illustrated; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030979#pone.0030979.s007" target="_blank">Fig. S7</a>). (D) <i>EGF signaling receptor expression.</i> To evaluate expression of erbB2 and EFGR1, together with a downstream effector of EGFR1, p-ERK1/2 (and total ERK1/2) in four primary tumors (DMBA D1, D18, D19, and D21), tumor tissue lysates (20 µgs) were compared with tumor tissue lysates from erbB2/neu transgenic mice (with 10.0, 4.0 and 2.0 µgs total protein), and with mammary gland from mid-pregnant and virgin mice. Vinculin was used as a loading control. Immunohistochemical staining for pEGFR in paraffin sections from normal mammary glands and tumors confirmed and extended the Western blotting results. Cell surface-associated pEGFR is typical of basal cells in normal mammary glands and this cell type-specific expression pattern is conserved in basaloid tumor cells. (Note that the green stain in the lumens is an artifact associated with sticky luminal secretions; panels C and D).</p

The basal cell fraction is relatively resistant to chemotherapeutics.

<p>(A) Tumor response to treatment with doxorubicin. Dissociated, second generation tumor cells (D19 shown here; results representative of 3 strains; 10 k cell inoculae) were iso-grafted into cleared fat pads, and the re-growth of tumors was observed by palpation and caliper measurement. Doxorubicin (single dose, 5 mgs/kg) was administered when tumors were 5 mm diameter (indicated as day 0), and this cohort compared to controls. Though initially substantially responsive to chemotherapy (2 days), after 2 weeks, tumor growth relapses. (B) <i>Selective arrest of luminal cells after exposure to chemotherapeutic</i>. The mitotic index (measured by anti-BrdU labeling, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030979#s4" target="_blank">Methods</a>) of basal (<b>K5</b>) and luminal (<b>K8</b>) cells was measured in untreated or doxorubicin-treated tumors (at the indicated times, either 2 days or 2 weeks after administration). (C–F) The fraction of dividing cells (BrdU-positive) per total population was measured during the treatment course (C, compare to growth rate of palpable tumor mass, panel A). The mitotic index for basal and luminal cell sub-populations is measured separately (D). The proportion of each cell subtype is shown with respect to the total population (E; grey are luminal, K8-positive, and black are basal, K5-positive; double negative cells (DN) stain for neither marker). The proportion of basal and luminal cells that make up the dividing cell population during drug treatment and recovery are shown in panel (F).</p

The tumor bi-lineal phenotype is stable over several generations and independent of tumor precursor cell.

<p>(A) Tumor re-growth in serial transplantation was assayed over time, by palpation (generating a Kaplan-Meier curve; n = sample number), inoculating with the cell numbers indicated. (B) Immunostaining of primary, secondary, and tertiary DTumors with anti-K5 (red) and anti-K8 (green) antisera. Scale bar = 50 µm. (C) The proportion of cells staining for each lineage marker <i>in vivo</i> (assayed from 3 separate tumors for each generation; 3 fields each) for each of the primary tumor and two serially regenerated tumors (Second T and Third T). (D) Corresponding immunocytochemical analysis and flow cytometric analysis of tumor cell populations from a primary tumor, for comparison with tumors regenerated from cell isografts of limiting dilutions of the cell type indicated (Lin⁻ and basal cell). Scale bar = 50 µm.</p

Purification of basal and luminal cell fractions from DMBA-induced mammary tumors.

<p>(A) Immunohistochemical stain of cell type specific markers (as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030979#pone-0030979-g001" target="_blank">Fig. 1</a>), and Ki67 (mitotic marker), in the following tissues prior to dissociation; normal 12 week-old mammary glands, a DTumor, and an MMTV-<i>neu</i> induced mammary tumor. Scale bar = 50 µm. (B) Flow cytometric separation of cells from BALB/c mammary glands and DTumors using immunophenotyping antibodies, EpCAM and CD49f. Mammary epithelial cell populations from normal glands and DTumors divided into two subpopulations, an EpCAM⁺/CD49f ^low/− luminal (L) population, and an EpCAM⁺/CD49f ^high basal (B) population. The specifics of flow cytometric analysis are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030979#pone.0030979.s005" target="_blank">Fig. S5</a>. (C) Immunostaining of the sorted cell fractions shown in Panel B was used to establish the relative purity of each “luminal” or “basal” cell fraction: K5 (blue), α-SMA (green), and K8 (red) (percentages are reported in the results). Scale bar = 50 µm. (D) Purified fractions of basal and luminal cells from either normal mammary gland or DTumors were cultured for 4 days and stained as for panel (C). Only basal cell fractions differentiate to both cell types (illustrating their bipotency). Compared to normal cells, tumor cells tend to co-express both keratin markers and lose their fate specification in cultures. Scale bar = 50 µm.</p