The P53 Independent Functions of Estrogen-Activated MDM2 in Cell Signaling and Mammary Architecture

Estrogen receptor positive (ER+) breast cancers often have MDM2 overexpression indicating a critical role for MDM2 in breast cancer tumorigenesis. The cancer genome atlas (TCGA) found that increased MDM2 expression is one of the four pathways that correlate with all breast cancer subtypes. MDM2 is mainly known as the negative regulator of wild type p53. However, aggressive breast cancers often have MDM2 overexpression and mutant p53 (mtp53). We previously reported that MDM2 provides an estrogen-mediated proliferative advantage to MCF-7 breast cancer cells (ER+, MDM2 overexpression, wild type p53), independent of wild type p53 in both 2D and 3D culture conditions. This and other studies suggest that MDM2 has a p53-independent role in tumorigenesis. To examine the estrogen-induced p53-independent roles of MDM2, we generated T47D breast cancer cells (ER+, MDM2 overexpression, mtp53 L194F) with shRNA to MDM2. As seen previously, estrogen treatment increased MDM2, and MDM2 knockdown did not change p53 protein levels. MDM2 knockdown inhibited estrogen mediated cell proliferation in 2D and 3D anchorage independent soft agar and matrigel culture. MDM2 knockdown decreased mass size, induced lumen formation and significantly reduced the number of phospho-histone H3 positive cells per mass indicating a decrease in mitotic rate. MDM2 knockdown also decreased Rb phosphorylation and E2F1 protein levels. Moreover, blocking estrogen signaling by estrogen antagonist Fulvestrant decreased MDM2 protein levels and phosphorylation of Rb. Our data place MDM2 as a central hub for estrogen-mediated p53-independent signal transduction. We demonstrate that MDM2 provides advantage to estrogen-induced breast cancer cell proliferation and disruption of mammary architecture through ER-MDM2-phosphoRb-E2F1 signaling pathway

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wildtype p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen- MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen- MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

<p>Abstract</p> <p>Background</p> <p>Genetic studies in <it>Drosophila melanogaster </it>reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways.</p> <p>Results</p> <p>Our biochemical studies show that insulin induces Myc protein accumulation in <it>Drosophila </it>S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3<it>β </it>) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3<it>β </it>activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia.</p> <p>Conclusions</p> <p>Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in <it>Drosophila</it>. At the biochemical level we found that both these pathways converge at GSK3<it>β </it>to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.</p

Directed Evolution of a Novel Heterochiral Ribonuclease Ribozyme and Kinetic Characterization of Heterochiral DNA Strand Displacement Reactions

Nature is inherently homochiral. L-DNA and L-RNA, the enantiomeric forms of native D-DNA and D-RNA respectively, do not occur naturally and are virtually bioorthogonal. Compared to common chemical modifications, L-DNA/RNA exhibit superior qualities such as increased biostability due to exceptional nuclease resistance, low immunogenicity, and minimal off-target effects. When interacting with an achiral physical or chemical environment, they behave similar to their native D-counterparts, facilitating easy optimization of designs based on L-DNA/RNA. However, while these “mirror-image” nucleic acids hybridize to each other, they are incapable of forming contiguous WC base-pairs with complementary native nucleic acids, a caveat that until recently precluded their use in applications at the interface of native biology. Redirecting the focus toward non-canonical heterochiral nucleic acid interactions based on non-WC hydrogen bonding, Van der Waals and hydrophobic interactions etc., overcomes this obstacle. This work focuses on exploring and expanding these novel recognition modalities with the aim of interfacing L-DNA/RNA with native nucleic acids using both structure and sequence-based approaches. Complex structural interactions in RNA govern almost all aspects of gene expression. Targeting structured RNA is thus integral to our fundamental understanding of RNA biology as well as in the development of therapeutics. Several heterochiral L-DNA/RNA aptamers have been evolved to bind their respective RNA targets with high specificity and affinity, in a structure-specific manner. In contrast, evolution of L-ribozymes is more challenging. RNA ligation and polymerization is the only reported example of heterochiral catalysis to date. In the first part of my thesis, I will discuss the in vitro evolution of a heterochiral ribonuclease ribozyme that interacts with a representative structured RNA target to mediate phosphodiester bond scission resulting in cleavage of the target. This opens a novel route to chemically target a specific RNA within its structural context while eliminating WC-based off-target hybridization. Furthermore, this approach is also promising for future therapeutic applications. The second part of this thesis focuses on sequence-specific interfacing strategies. Our research group recently described a novel technique called “heterochiral” DNA strand displacement reactions that utilize an achiral peptide nucleic acid (PNA) mediator to exchange sequence information between D-DNA and L-DNA, thus providing a route to exploit the advantageous properties of L-DNA in dynamic DNA nanotechnology applications. In this work I present extensive kinetic characterization of these novel reactions by systematically varying key design parameters in order to establish a set of design principles that will facilitate the rational design of such devices for biomedical applications in the future. Additionally, investigation of the biophysical mechanism of these reactions reveals a novel stereochemical control over reaction kinetics, that adds to the versatility of future designs

Lymphangioleiomyomatosis: A Metastatic Lung Disease

Lymphangioleiomyomatosis (LAM) is a rare disease affecting women, caused by somatic mutations in the TSC1 or TSC2 genes, and driven by estrogen. Similar to many cancers, it is metastatic, primarily to the lung. Despite its monogenetic nature, like many cancers, LAM is a heterogeneous disease. The cellular constituents of LAM are very diverse, including mesenchymal, epithelial, endothelial, and immune cells. LAM is characterized by dysregulation of many cell signaling pathways, distinct populations of LAM cells, and a rich microenvironment, in which the immune system appears to play an important role. This review delineates the heterogeneity of LAM and focuses on the metastatic features of LAM, the deregulated signaling mechanisms and the tumor microenvironment. Understanding the tumor-host interaction in LAM may provide insights into the development of new therapeutic strategies, which could be combinatorial or superlative to Sirolimus, the current U.S. Food and Drug Administration-approved treatment

The effect of intermediate layer on synthesis and gas permeation properties of NaA zeolite membrane

NaA zeolite membrane coating was successfully synthesized on an alumina porous disc by hydrothermal treatment. The effects of synthesis parameters, such as seeding condition (in situ, ex situ), synthesis time, synthesis stages, application of intermediate layer, etc., on membrane characteristics were investigated. Surface seeding accelerates the zeolite crystallization process on the support surface, and also enhances the formation of homogeneous NaA zeolite layer. But the main problem associated with membrane coating synthesis is crack formation. Formation of crack was reduced by applying intermediate layer, between the support surface and seed layer. A thin Boehmite layer was applied to the support surface before applying seed crystals to enhance the adherence between zeolite seed layer and boehmite layer by hydrogen bonding and also to increase the mechanical strength of the membrane layer. The quality of the membrane layer can be improved by employing the multi-stage coating methods. The permeance of O(2), N(2) decreased as kinetic diameter of gases increased, which shows the molecular sieving effect of the NaA membrane. The permselectivity of O(2)/N(2) was 1.9-2.0. This value of permselectivity ratio is higher than Knudsen diffusion ratio 0.94; it was also confirmed the molecular sieving properties of synthesized NaA zeolite membrane