Sridaran Natesan

Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial–mesenchymal transition

The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor–stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR–stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial–mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal–related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR–overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor–stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

Human and Drosophila Homeodomain Proteins That Enhance the DNA-Binding Activity of Serum Response Factor

Cells with distinct developmental histories can respond differentially to identical signals, suggesting that signals are interpreted in a fashion that reflects a cells identity. How this might occur is suggested by the observation that proteins of the homeodomain family, including a newly identified human protein, enhance the DNA-binding activity of serum response factor, a protein required for the induction of genes by growth and differentiation factors. Interaction with proteins of the serum response factor family may allow homeodomain proteins to specify the transcriptional response to inductive signals. Moreover, because the ability to enhance the binding of serum response factor to DNA resides within the homeodomain but is independent of homeodomain DNA-binding activity, this additional activity of the homeodomain may account for some of the specificity of action of homeodomain proteins in development.

Proteasome‐mediated degradation of transcriptional activators correlates with activation domain potency in vivo

We show that the intracellular concentration of transcriptional activator proteins is regulated by the proteasome‐mediated protein degradation pathway. The rate of degradation of activators by proteasomes correlates with activation domain potency in vivo. Mutations either in the activation domain residues involved in target protein interaction or in the DNA‐binding domain residues essential for DNA binding abolish the transcriptional activation function in vivo and render the activator resistant to degradation by proteasomes. Finally, using a rapamycin‐regulated gene expression system, we show that recruiting activation domains to DNA‐bound receptor proteins greatly enhanced the rate of degradation of reconstituted activators. These observations suggest that in mammalian cells efficient recruitment of activator–target protein complexes to the promoter means that they are subjected to rapid degradation by proteasomes. We propose that proteasome‐mediated control of the intracellular levels of transcriptional activators could play an important role in the regulation of gene expression.

Complementary patterns of gene expression by human oligodendrocyte progenitors and their environment predict determinants of progenitor maintenance and differentiation

Glial progenitor cells are abundant in adult human white matter. This study was designed to identify signaling pathways regulating their self‐renewal and fate.

YY1 facilitates the association of serum response factor with the c-fos serum response element.

YY1 is a multifunctional transcription factor that acts as an activator or repressor in different contexts. YY1 binds to multiple sites in the mouse c-fos promoter, inducing at each site a sharp DNA bend. Binding of YY1 to a site situated between the cyclic AMP response element (CRE) and the TATA box bends the DNA in a way that interferes with the interaction of proteins bound at the CRE and TATA elements, resulting in repression of transcription. Here, we show that binding of YY1 to a different site in the c-fos promoter has a different result. Binding of YY1 to the c-fos serum response element (SRE) enhances the binding of serum response factor (SRF). This enhancement requires the binding of YY1 to SRE DNA. YY1 and SRF can cooccupy the SRE at least transiently. In the region of overlapping contact, YY1 contacts DNA in the major groove, while SRF contacts DNA in the minor groove. YY1 also enhances the association of SRF with the SRE in transfected insect cells. Thus, although YY1 induces similar structural changes in DNA at different binding sites, it can have distinct local effects on protein-DNA and protein-protein interactions. These data support a general role for YY1 in the building of highly organized promoter complexes.

PTK6 Regulates IGF-1-Induced Anchorage-Independent Survival

Background Proteins that are required for anchorage-independent survival of tumor cells represent attractive targets for therapeutic intervention since this property is believed to be critical for survival of tumor cells displaced from their natural niches. Anchorage-independent survival is induced by growth factor receptor hyperactivation in many cell types. We aimed to identify molecules that critically regulate IGF-1-induced anchorage-independent survival. Methods and Results We conducted a high-throughput siRNA screen and identified PTK6 as a critical component of IGF-1 receptor (IGF-1R)-induced anchorage-independent survival of mammary epithelial cells. PTK6 downregulation induces apoptosis of breast and ovarian cancer cells deprived of matrix attachment, whereas its overexpression enhances survival. Reverse-phase protein arrays and subsequent analyses revealed that PTK6 forms a complex with IGF-1R and the adaptor protein IRS-1, and modulates anchorage-independent survival by regulating IGF-1R expression and phosphorylation. PTK6 is highly expressed not only in the previously reported Her2+ breast cancer subtype, but also in high grade ER+, Luminal B tumors and high expression is associated with adverse outcomes. Conclusions These findings highlight PTK6 as a critical regulator of anchorage-independent survival of breast and ovarian tumor cells via modulation of IGF-1 receptor signaling, thus supporting PTK6 as a potential therapeutic target for multiple tumor types. The combined genomic and proteomic approaches in this report provide an effective strategy for identifying oncogenes and their mechanism of action.

Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes.

Glial progenitor cells (GPCs) are a potential source of malignant gliomas. We used A2B5-based sorting to extract tumorigenic GPCs from human gliomas spanning World Health Organization grades II-IV. Messenger RNA profiling identified a cohort of genes that distinguished A2B5+ glioma tumor progenitor cells (TPCs) from A2B5+ GPCs isolated from normal white matter. A core set of genes and pathways was substantially dysregulated in A2B5+ TPCs, which included the transcription factor SIX1 and its principal cofactors, EYA1 and DACH2. Small hairpin RNAi silencing of SIX1 inhibited the expansion of glioma TPCs in vitro and in vivo, suggesting a critical and unrecognized role of the SIX1-EYA1-DACH2 system in glioma genesis or progression. By comparing the expression patterns of glioma TPCs with those of normal GPCs, we have identified a discrete set of pathways by which glial tumorigenesis may be better understood and more specifically targeted.

Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Structure and Function of Glucose Binding Protein–Single Walled Carbon Nanotube Complexes

Understanding the structure and function of glucose binding proteins (GBP) complexed with single walled carbon nanotubes (SWNTs) is important for the development of applications including fluorescent sensors and nanostructure particle tracking. Herein, circular dichroism (CD), thermal denaturation, photo-absorption spectroscopy and atomic force microscopy are used to study these nanostructures. The protein retains its glucose-binding activity after complexation and is thermally stable below 36 °C. However, the SWNT lowers the midpoint denaturation temperature (Tm) by 5 °C and 4 °C in the absence and presence of 10 mM glucose, respectively. This data highlights that using techniques such as CD and thermal denaturation may be necessary to fully characterize such protein-nanomaterial nanostructures.

A Niche-Defying Feat: Induced Oligoneogenesis in the Adult Dentate Gyrus

In a recent paper in Nature Neuroscience, Jessberger et al. (2008) report that overexpression of ASCL1 (mash1) directs adult hippocampal progenitors to adopt an oligodendrocytic fate. The effect is specific to the hippocampal niche in vivo, indicating that cell-autonomous and niche-defined factors collaborate to instruct cell fate choices.