Jennifer Liu

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors.

Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-κB site on its promoter

We report here that human monocytic/macrophage THP-1 cells express the neurokinin 1 receptor (NK-1R), and that exposure of these cells to the proinflammatory cytokine IL-1β increased the expression of the NK-1R gene at the mRNA and protein levels. Because IL-1β function involves nuclear factor κB (NF-κB) activation, these data suggest that this increase in the expression of the NK-1R gene is mediated by the NF-κB transcription factor. An earlier report noted that the promoter region of the human NK-1R gene contains a putative binding site for NF-κB [Takahashi, K., Tanaka, A., Hara, M. & Nakanishi, S. (1992) Eur. J. Biochem. 204, 1025–1033]. Here we demonstrate that this is indeed a functional NF-κB-binding site, and that NF-κB is responsible for regulating the expression of the NK-1R gene by binding to the promoter region of the NK-1R gene. To further substantiate that the observed NF-κB-dependent IL-1β induction of the human NK-1R gene is regulated via a transcriptional event through this NF-κB site on the NK-1R gene promoter, we transfected THP-1 cells with a luciferase promoter-reporter construct containing the 5′ promoter region of the human NK-1R gene. Exposure of these cells to IL-1β or overexpression of NF-κB cDNAs resulted in a significant increase in the amount of luciferase activity that was diminished greatly in cells transfected with IκBα, the NF-κB inhibitor. These results directly implicate NF-κB in the regulation of the NK-1R gene and provide a molecular mechanism for the increase in expression of the NK-1R gene in responsive cells.

Chemically programmed cell adhesion with membrane-anchored oligonucleotides

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane-anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, and tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells that are chemically immobilized on a passivated glass surface.

Directing the assembly of spatially organized multicomponent tissues from the bottom up

The complexity of the human body derives from numerous modular building blocks assembled hierarchically across multiple length scales. These building blocks, spanning sizes ranging from single cells to organs, interact to regulate development and normal organismal function but become disorganized during disease. Here, we review methods for the bottom-up and directed assembly of modular, multicellular, and tissue-like constructs in vitro. These engineered tissues will help refine our understanding of the relationship between form and function in the human body, provide new models for the breakdown in tissue architecture that accompanies disease, and serve as building blocks for the field of regenerative medicine.

A naturally occuring insertion of a single amino acid rewires transcriptional regulation by glucocorticoid receptor isoforms

Significance For proteins to be able to have context-specific activities, they can adopt context-specific conformations that enhance or restrict their activity. For transcriptional regulatory factors, such a context-specific signal is provided by the sequence of the DNA response element to which it binds. Here we show how one signal, an alternative splicing event, rewires a transcriptional regulatory protein to respond differently to a second signal, the DNA sequence to which it binds, by changing the functional interplay between protein domains. Together, our findings argue that bidirectional allosteric signaling between the DNA:protein interface and other regulatory domains fine tunes the activity of transcriptional regulatory factors toward individual target genes. In addition to guiding proteins to defined genomic loci, DNA can act as an allosteric ligand that influences protein structure and activity. Here we compared genome-wide binding, transcriptional regulation, and, using NMR, the conformation of two glucocorticoid receptor (GR) isoforms that differ by a single amino acid insertion in the lever arm, a domain that adopts DNA sequence-specific conformations. We show that these isoforms differentially regulate gene expression levels through two mechanisms: differential DNA binding and altered communication between GR domains. Our studies suggest a versatile role for DNA in both modulating GR activity and also in directing the use of GR isoforms. We propose that the lever arm is a ”fulcrum” for bidirectional allosteric signaling, conferring conformational changes in the DNA reading head that influence DNA sequence selectivity, as well as conferring changes in the dimerization domain that connect functionally with remote regulatory surfaces, thereby influencing which genes are regulated and the magnitude of their regulation.

Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

All mixed up: defining roles for β-cell subtypes in mature islets

Following differentiation during fetal development, β cells further adapt to their postnatal role through functional maturation. While adult islets are thought to contain functionally mature β cells, recent analyses of transgenic rodent and human pancreata reveal a number of novel heterogeneity markers in mammalian β cells. The marked heterogeneity long after maturation raises the prospect that diverse populations harbor distinct roles aside from glucose-stimulated insulin secretion. In this review, we outline our current understanding of the β-cell maturation process, emphasize recent literature on novel heterogeneity markers, and offer perspectives on reconciling the findings from these two areas.

CHAPTER 38 – Psychological Aspects of Ovarian Cancer and BRCA Testing

This chapter highlights psychosocial issues related to the diagnosis, management, and palliative care of ovarian cancer. The chapter discusses common psychiatric diagnoses of anxiety, depression, and delirium. Furthermore, the chapter reviews the psychiatric impact of genetic testing and the doctor-patient relationship. Cancer disrupts all aspects of life. Each persons concept of cancer, death, and dying varies depending on the persons societal, philosophical, spiritual, and religious beliefs. Ovarian cancer patients often face difficult physical symptoms and psychological problems throughout their clinical course. Approximately one-third of patients with newly diagnosed or recurrent cancer experience psychological distress. The doctor-patient relationship is vital in helping the patient with ovarian cancer and negotiating the challenges to the family. The team of nurses, social workers, mental health professionals, and community resources are crucial in providing the optimal emotional support. Symptoms of anxiety and depression are usually experienced by significant number of patients with cancer, either due to the direct effects of the cancer, treatments given for the cancer, or as a manifestation of the psychological distress experienced in response to diagnosis of cancer. These symptoms should be managed, in consultation with a psychiatrist providing the appropriate pharmacological and psychotherapeutic support.