Antonis Kourtidis

Computational Identification of a p38SAPK-Regulated Transcription Factor Network Required for Tumor Cell Quiescence

The stress-activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools, we identified a transcription factor (TF) network regulated by p38alpha/beta and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Furthermore, induction of p53 by p38 was dependent on c-Jun down-regulation. Accordingly, RNAi down-regulation of BHLHB3 or p53 interrupted tumor cell quiescence, while down-regulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers.

Dual Function of Pancreatic Endoplasmic Reticulum Kinase in Tumor Cell Growth Arrest and Survival

Pancreatic endoplasmic reticulum kinase (PERK)-eIF2 alpha signaling, a component of the endoplasmic reticulum (ER) stress response, has been proposed as a therapeutic target due to its importance to cell survival in hypoxic tumors. In this study, we show that in addition to promoting survival, PERK can also suppress tumor growth of advanced carcinomas. Our results show that in squamous carcinoma T-HEp3 cells, which display low PERK-eIF2 alpha signaling, inducible activation of an Fv2E-PERK fusion protein results in a strong G(0)-G(1) arrest in vitro. Most importantly, Fv2E-PERK activation, in addition to promoting survival in vitro, inhibits T-HEp3 and SW620 colon carcinoma growth in vivo. Increased PERK activation is linked to enhanced p-eIF2 alpha levels, translational repression, and a decrease in Ki67, pH 3, and cycD1/D3 levels, but not to changes in angiogenesis or apoptosis. Experimental reduction of PERK activity, or overexpression of GADD34 in a spontaneously arising in vivo quiescent variant of HEp3 cells that displays strong basal PERK-eIF2 alpha activation, reverts their quiescent phenotype. We conclude that the growth-inhibitory function of PERK is preserved in tumors and upon proper reactivation can severely inhibit tumor growth through induction of quiescence. This is an important consideration in the development of PERK-based therapies, as its inhibition may facilitate the proliferation of slow-cycling or dormant tumor cells.

p120 Catenin: An Essential Regulator of Cadherin Stability, Adhesion-Induced Signaling, and Cancer Progression

p120 catenin is the best studied member of a subfamily of proteins that associate with the cadherin juxtamembrane domain to suppress cadherin endocytosis. p120 also recruits the minus ends of microtubules to the cadherin complex, leading to junction maturation. In addition, p120 regulates the activity of Rho family GTPases through multiple interactions with Rho GEFs, GAPs, Rho GTPases, and their effectors. Nuclear signaling is affected by the interaction of p120 with Kaiso, a transcription factor regulating Wnt-responsive genes as well as transcriptionally repressing methylated promoters. Multiple alternatively spliced p120 isoforms and complex phosphorylation events affect these p120 functions. In cancer, reduced p120 expression correlates with reduced E-cadherin function and with tumor progression. In contrast, in tumor cells that have lost E-cadherin expression, p120 promotes cell invasion and anchorage-independent growth. Furthermore, p120 is required for Src-induced oncogenic transformation and provides a potential target for future therapeutic interventions.

VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx

VEGF causes translocation of Syx from endothelial cell junctions, promoting junction disassembly, whereas Angtiopoietin-1 maintains Syx at the junctions and stabilizes them.

An RNA Interference Screen Identifies Metabolic Regulators NR1D1 and PBP as Novel Survival Factors for Breast Cancer Cells with the ERBB2 Signature

Overexpression of the adverse prognostic marker ERBB2 occurs in 30% of breast cancers; however, therapies targeting this gene have not proved to be as effective as was initially hoped. Transcriptional profiling meta-analyses have shown that there are approximately 150 genes co-overexpressed with ERBB2, suggesting that these genes may represent alternative factors influencing ERBB2-positive tumors. Here we describe an RNA interference-based analysis of these genes that identifies transcriptional regulators of fat synthesis and storage as being critical for the survival of these cells. These transcription factors, nuclear receptor subfamily 1, group D, member 1 (NR1D1) and peroxisome proliferator activated receptor gamma binding protein (PBP), both reside on ERBB2-containing 17q12-21 amplicons and are part of the ERBB2 expression signature. We show that NR1D1 and PBP act through a common pathway in upregulating several genes in the de novo fatty acid synthesis network, which is highly active in ERBB2-positive breast cancer cells. Malate dehydrogenase 1 and malic enzyme 1, enzymes that link glycolysis and fatty acid synthesis, are also regulated by NR1D1. The resulting high-level fat production from increased expression of these genes likely contributes to an abnormal cellular energy metabolism based on aerobic glycolysis. Together, these results show that the cells of this aggressive form of breast cancer are genetically preprogrammed to depend on NR1D1 and PBP for the energy production necessary for survival.

The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.

Peroxisome proliferator-activated receptor-γ protects ERBB2-positive breast cancer cells from palmitate toxicity

IntroductionAccumulation of fatty acids and neutral lipids in nonadipose tissues is cytotoxic. We recently showed that ERBB2-positive breast cancer cells produce significantly high amounts of fats, because of overexpression of the peroxisome proliferator-activated receptor (PPAR)γ-binding protein and the nuclear receptor NR1D1 (nuclear receptor subfamily 1, group D, member 1; Rev-erbα). These genes upregulate de novo fatty acid synthesis, which is a critical pathway for the energy production and survival of these cells. NR1D1 and PPARγ-binding protein are functionally related to PPARγ, a well established positive regulator of adipogenesis and lipid storage.MethodsThe effects of GW9662 and exogenously added palmitate on breast cells (BT474, MDA-MB-361, MCF-7, and human mammary epithelial cells) in monolayer culture were assessed. Mass spectrometric quantitation of fatty acids and fluorescence-based high content microscopy assays of cell growth, apoptosis, triglyceride storage and reactive oxygen species production were used.ResultsERBB2-positive breast cancer cells are more sensitive to inhibition of PPARγ activity by the antagonist GW9662. PPARγ inhibition results in increased levels of total fats in the cells, mostly because of increased amounts of palmitic and stearic unsaturated acids. Administration of exogenous palmitate is lethal to ERBB2-positive but not to ERBB2-negative cells. GW9662 exacerbates the effects of palmitate addition on BT474 and MDA-MB-361 cells, but it has no significant effect on MCF-7 and human mammary epithelial cells. Palmitate administration results in a fivefold to tenfold greater increase in fat stores in ERBB2-negative cells compared with ERBB2-positive cells, which suggests that the ERBB2-positive cells have maximized their ability to store fats and that additional palmitate is toxic to these cells. Both PPARγ inhibition and palmitate administration result in increased reactive oxygen species production in BT474 cells. The cell death that results from this treatment can be counteracted by the antioxidant N-acetyl cysteine.ConclusionsOur findings indicate that PPARγ activity enables ERBB2-positive breast cancer cells, which produce high levels of fat, to convert fatty acids to triglycerides, allowing these cells to avert the cell death that results from lipotoxicity. Endogenous palmitate toxicity represents a genetically based property of ERBB2-positive breast cancer that can be exploited for therapeutic intervention.

MicroRNA target detection and analysis for genes related to breast cancer using MDLcompress

We describe initial results of miRNA sequence analysis with the optimal symbol compression ratio (OSCR) algorithm and recast this grammar inference algorithm as an improved minimum description length (MDL) learning tool: MDLcompress. We apply this tool to explore the relationship between miRNAs, single nucleotide polymorphisms (SNPs), and breast cancer. Our new algorithm outperforms other grammar-based coding methods, such as DNA Sequitur, while retaining a two-part code that highlights biologically significant phrases. The deep recursion of MDLcompress, together with its explicit two-part coding, enables it to identify biologically meaningful sequence without needlessly restrictive priors. The ability to quantify cost in bits for phrases in the MDL model allows prediction of regions where SNPs may have the most impact on biological activity. MDLcompress improves on our previous algorithm in execution time through an innovative data structure, and in specificity of motif detection (compression) through improved heuristics. An MDLcompress analysis of 144 over expressed genes from the breast cancer cell line BT474 has identified novel motifs, including potential microRNA (miRNA) binding sites that are candidates for experimental validation.

A central role for cadherin signaling in cancer

Cadherins are homophilic adhesion molecules with important functions in cell-cell adhesion, tissue morphogenesis, and cancer. In epithelial cells, E-cadherin accumulates at areas of cell-cell contact, coalesces into macromolecular complexes to form the adherens junctions (AJs), and associates via accessory partners with a subcortical ring of actin to form the apical zonula adherens (ZA). As a master regulator of the epithelial phenotype, E-cadherin is essential for the overall maintenance and homeostasis of polarized epithelial monolayers. Its expression is regulated by a host of genetic and epigenetic mechanisms related to cancer, and its function is modulated by mechanical forces at the junctions, by direct binding and phosphorylation of accessory proteins collectively termed catenins, by endocytosis, recycling and degradation, as well as, by multiple signaling pathways and developmental processes, like the epithelial to mesenchymal transition (EMT). Nuclear signaling mediated by the cadherin associated proteins β-catenin and p120 promotes growth, migration and pluripotency. Receptor tyrosine kinase, PI3K/AKT, Rho GTPase, and HIPPO signaling, are all regulated by E-cadherin mediated cell-cell adhesion. Finally, the recruitment of the microprocessor complex to the ZA by PLEKHA7, and the subsequent regulation of a small subset of miRNAs provide an additional mechanism by which the state of epithelial cell-cell adhesion affects translation of target genes to maintain the homeostasis of polarized epithelial monolayers. Collectively, the data indicate that loss of E-cadherin function, especially at the ZA, is a common and crucial step in cancer progression.

TEM4 is a junctional Rho GEF required for cell-cell adhesion, monolayer integrity and barrier function

Summary Signaling events mediated by Rho family GTPases orchestrate cytoskeletal dynamics and cell junction formation. The activation of Rho GTPases is tightly regulated by guanine-nucleotide-exchange factors (GEFs). In this study, we identified a novel Rho-specific GEF called TEM4 (tumor endothelial marker 4) that associates with multiple members of the cadherin–catenin complex and with several cytoskeleton-associated proteins. Depending on confluence, TEM4 localized to either actin stress fibers or areas of cell–cell contact. The junctional localization of TEM4 was independent of actin binding. Depletion of endogenous TEM4 by shRNAs impaired Madin–Darby canine kidney (MDCK) and human umbilical vein endothelial cell (HUVEC) cell junctions, disrupted MDCK acini formation in 3D culture and negatively affected endothelial barrier function. Taken together, our findings implicate TEM4 as a novel and crucial junctional Rho GEF that regulates cell junction integrity and epithelial and endothelial cell function.