Elizabeth H. Williams

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

L-ascorbic acid (vitamin C) is a powerful reducing agent found in millimolar concentrations in plants, and is proposed to play an important role in scavenging free radicals in plants and animals. However, surprisingly little is known about the role of this antioxidant in plant environmental stress adaptation or ascorbate biosynthesis. We report the isolation of soz1, a semi-dominant ozone-sensitive mutant that accumulates only 30% of the normal ascorbate concentration. The results of genetic approaches and feeding studies show that the ascorbate concentration affects foliar resistance to the oxidizing gas ozone. Consistent with the proposed role for ascorbate in reactive oxygen species detoxification, lipid peroxides are elevated in soz1, but not in wild type following ozone fumigation. We show that the soz1 mutant is hypersensitive to both sulfur dioxide and ultraviolet B irradiation, thus implicating ascorbate in defense against varied environmental stresses. In addition to defining the first ascorbate deficient mutant in plants, these results indicate that screening for ozone-sensitive mutants is a powerful method for identifying physiologically important antioxidant mechanisms and signal transduction pathways. Analysis of soz1 should lead to more information about the physiological roles and metabolism of ascorbate.

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in Drosophila have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes in vivo and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1alpha in Hh pathway activation.