The contribution of basal and luminal cells to cancer progression and metastasis is poorly understood. We report generation of reporter systems driven by either keratin-14 (K14) or keratin-8 (K8) ...promoter that not only express a fluorescent protein but also an inducible suicide gene. Transgenic mice express the reporter genes in the right cell compartments of mammary gland epithelia and respond to treatment with toxins. In addition, we engineered the reporters into 4T1 metastatic mouse tumor cell line and demonstrate that K14+ cells, but not K14- or K8+, are both highly invasive in three-dimensional (3D) culture and metastatic in vivo. Treatment of cells in culture, or tumors in mice, with reporter-targeting toxin inhibited both invasive behavior and metastasis in vivo. RNA sequencing (RNA-seq), secretome, and epigenome analysis of K14+ and K14- cells led to the identification of amphoterin-induced protein 2 (Amigo2) as a new cell invasion driver whose expression correlated with decreased relapse-free survival in patients with TP53 wild-type (WT) breast cancer.
There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of ...human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.
The polarity protein Scribble (SCRIB) regulates apical-basal polarity, directional migration and tumour suppression in Drosophila and mammals. Here we report that SCRIB is an important regulator of ...myeloid cell functions including bacterial infection and inflammation. SCRIB interacts directly with the NADPH oxidase (NOX) complex in a PSD95/Dlg/ZO-1 (PDZ)-domain-dependent manner and is required for NOX-induced reactive oxygen species (ROS) generation in culture and in vivo. On bacterial infection, SCRIB localized to phagosomes in a leucine-rich repeat-dependent manner and promoted ROS production within phagosomes to kill bacteria. Unexpectedly, SCRIB loss promoted M1 macrophage polarization and inflammation. Thus, SCRIB uncouples ROS-dependent bacterial killing activity from M1 polarization and inflammatory functions of macrophages. Modulating the SCRIB-NOX pathway can therefore identify ways to manage infection and inflammation with implications for chronic inflammatory diseases, sepsis and cancer.
Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead ...to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies.
Three sequential methylations of phosphoethanolamine (PEA) are required for the synthesis of phosphocholine (PCho) in plants. A cDNA encoding an N-methyltransferase that catalyzes the last two ...methylation steps was cloned from Arabidopsis by heterologous complementation of a Saccharomyces cerevisiae cho2, opi3 mutant. The cDNA encodes phosphomethylethanolamine N-methyltransferase (PMEAMT), a polypeptide of 475 amino acids that is organized as two tandem methyltransferase domains. PMEAMT shows 87% amino acid identity to a related enzyme, phosphoethanolamine N-methyltransferase, an enzyme in plants that catalyzes all three methylations of PEA to PCho. PMEAMT cannot use PEA as a substrate, but assays using phosphomethylethanolamine as a substrate result in both phosphodimethylethanolamine and PCho as products. PMEAMT is inhibited by the reaction products PCho and S-adenosyl-l-homocysteine, a property reported for phosphoethanolamine N-methyltransferase from various plants. An Arabidopsis mutant with a T-DNA insertion associated with locus At1g48600 showed no transcripts encoding PMEAMT. Shotgun lipidomic analyses of leaves of atpmeamt and wild-type plants generated phospholipid profiles showing the content of phosphatidylmethylethanolamine to be altered relative to wild type with the content of a 34:3 lipid molecular species 2-fold higher in mutant plants. In S. cerevisiae, an increase in PtdMEA in membranes is associated with reduced viability. This raises a question regarding the role of PMEAMT in plants and whether it serves to prevent the accumulation of PtdMEA to potentially deleterious levels.
Using spectral filters, Cladonia uncialis and Cladina rangiferina were exposed for seven days to visible light supplemented with two intensities of three different bands of ultraviolet in a growth ...chamber. The concentrations of lichen secondary products separated by thin-layer chromatography were then determined densitometrically. In Cladonia uncialis, higher intensity exposure to visible light enhanced with UV-A, while UV-B and UV-C were excluded, significantly increased usnic acid concentrations over control levels. However, less usnic acid accumulated under high intensity enhancement of both UV-A and UV-B. The effect of supplemental UV-A and UV-B was the same on atranorin concentrations in Cladina rangiferina. This suggested that UV-A caused increased accumulation of both compounds, but that the effect was negated by UV-B. Concentrations of usnic acid in natural populations of Cladina mitis were significantly lower in spring and summer when UV-B flux is greatest.
The cell polarity protein scribble (SCRIB) is a crucial regulator of polarization, cell migration and tumorigenesis. Whereas SCRIB is known to regulate early stages of mouse mammary gland ...development, its function in the adult gland is not known. Using an inducible RNA interference (RNAi) mouse model for downregulating SCRIB expression, we report an unexpected role for SCRIB as a positive regulator of cell proliferation during pregnancy-associated mammary alveologenesis. SCRIB was required in the epithelial cell compartment of the mammary gland. Lack of SCRIB attenuated prolactin-induced activation of the JAK2-STAT5 signaling pathway. In addition, loss of SCRIB resulted in the downregulation of prolactin receptor (PRLR) at cell surface and its accumulation in intracellular structures that express markers of the Golgi complex and the recycling endosome. Unlike its role in virgin gland as a negative regulator cell proliferation, SCRIB is a positive regulator of mammary epithelial cell proliferation during pregnancy.
A densitometric technique was developed that permitted rapid quantification of lichen compounds separated by thin-layer chromatography. Thin-layer plates were scanned in a densitometer using a UV ...light source and density-dependent absorbance or fluorescence was measured for each compound. The lichen products tested differed with respect to photostability under UV, and thus in the time taken to achieve constant densitometric readings. Minimal exposure times required for stabilization of the compounds tested ranged from 3 min to 2.5 hr.
In plants, phosphocholine (PCho) is a precursor to the membrane component phosphatidylcholine (PtdCho) and free choline (Cho). A mutant Saccharomyces cerevisiae yeast strain unable to produce PtdCho ...without exogenous choline was used for transformation with an Arabidopsis cDNA library cloned in the yeast expression vector pFK61. A plant cDNA associated with locus At1g48600 functionally complemented the mutant by restoring growth on minimal synthetic medium lacking choline but containing the phosphobase phosphomethylethanolamine (PMEA). Crude extracts prepared from the yeast showed a novel capacity to convert PMEA to phosphodimethylethanolamine (PDEA) and PCho and hence this enzyme has been named Arabidopsis S-adenosyl-L-methionine (AdoMet): phosphomethylethanolamine N-methyltransferase (AtPMEAMT).
AtPMEAMT is a bipartite enzyme containing tandem N-and C-terminal AdoMet-binding domains. The predicted amino acid sequence shows an 87% identity to the previously characterized AdoMet: phosphoethanolamine N-methyltransferase (AtPEAMT) from Arabidopsis. An important distinction between AtPMEAMT and AtPEAMT is that the former enzyme is unable to methylate phosphoethanolamine (PEA). However, both AtPEAMT and AtPMEAMT can methylate PMEA and PDEA, two phosphobase intermediates ofPCho synthesis. The apparent Km values were determined for AtPEAMT and AtPMEAMT toward PMEA and PDEA and found to be 0.32 and 0.14 mM, respectively, for PEAMT and 0.16 and 0.03 mM, respectively, for PMEAMT. The N-and C-terminal Ado Met-domains of PEAMT and PMEAMT were cloned separately into a pET30a(+) vector for protein expression and extracts containing recombinant proteins were assayed for phosphobase methyltransferase activity. Only the gene product encoding the domain associated with the C-terminal half of PMEAMT methylated both PMEA and PDEA, an activity found with the native protein. A chimera was produced by combining the N-terminal half ofPEAMT and the C-terminal half of PMEAMT. The chimeric protein is able to methylate PEA, PMEA and PDEA indicating that a feature associated with the N-terminal half of PEAMT is required for PEA methylation. This result suggests that differences associated with the N-terminal domain are likely responsible for the inability ofPMEAMT to use PEA as a substrate.
An Arabidopsis mutant line with a T-DNA insertion in the promoter region of PMEAMT (SALK 006037) was obtained and RT-PCR analysis of plants homozygous for the insert showed that the mutant lacks transcripts associated with this gene. Relative to wild-type plants grown under identical conditions the mutant plants showed no visible difference in morphological or developmental phenotype. However, shotgun lipidomics using electrospray ionization tandem mass spectrometry showed a 2.1-fold greater abundance ofa 34:3 phosphatidylmethylethanolamine (PtdMEA) molecular species in mutant plants compared to wild-type. One biological role of PMEAMT may be to reduce the likelihood for PtdMEA incorporation into phospholipids ofmembranes. PtdMEA incorporation in membranes is associated with reduced viability of yeast but its effect on the physiology ofplants is, as yet, unknown.
Thesis
Doctor of Philosophy (PhD)
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