Department of Cell and Developmental Biology

Since mitochondrial dysfunction plays an important role in the pathogenesis of dopaminergic neurodegeneration in Parkinson's disease, we determined the expression of genes related to mitochondrial function in the substantia nigra of mice treated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) using a cDNA array. MPTP treatment significantly depleted striatal dopamine, but did not result in apparent neuronal loss in the substantia nigra at 3 and 18 days post-treatment. We also examined changes in genes in the hypothalamus, a region containing dopaminergic neurons that are relatively resistant to MPTP. Finally, we confirmed those genes identified by microarrays as differentially expressed in the substantia nigra but not in the hypothalamus using in situ hybridization. Our results demonstrated that MPTP significantly changed the expressions of six genes in nigral neurons, four of which were related to the mitochondrial electron transport chain: the NADH-ubiquinone oxidoreductase 13 kDa B subunit, the NADH-ubiquinone oxidoreductase MNLL subunit, cytochrome c, and the cytochrome c oxidase Va subunit. Two other differentially expressed genes were the dihydropyridine-sensitive L-type calcium channel a-2 subunit precursor and type III a-1 procollagen. None of these six genes are encoded by mitochondrial DNA. The potential significance of these gene alterations in the context of Parkinson's disease is discussed.

Although both amphiregulin and TGF-α are known to exert their effects through the EGF receptor, we find that concentrations of recombinant human amphiregulin and TGF-α that are equipotent in EGF receptor activation and mitogenesis exhibit markedly different effects on MDCK cell morphology. Amphiregulin induces a spindle-like morphology that is associated with a redistribution of E-cadherin from a Triton-insoluble to Triton-soluble

Activating mutations of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis. Constitutive activation of the three canonical family members, KRAS, NRAS, and HRAS segregate strongly by tissue type. Of these, KRAS mutations predominate in human tumors, including those arising from the colon and lung. We sought to compare the oncogenic contributions of different RAS isoforms in a comparable genetic setting and to explore downstream molecular changes that may explain the apparent differential oncogenic effects of the various RAS family members. We utilized colorectal cancer cell lines characterized by oncogenic KRAS in parallel with isogenically derived lines in which the mutant allele has been disrupted. We additionally attempted to reconstitute the isogenic derivatives with oncogenic forms of other RAS family members and analyze them in parallel. Pairwise analysis of HCT 116 and DLD-1 cell lines as well as their isogenic derivatives reveals distinct K-RAS(G13D) signatures despite the genetic similarities of these cell lines. In DLD-1, for example, oncogenic K-RAS enhances the motility of these cells by downregulation of Rap1 activity, yet is not associated with increased ERK1/2 phosphorylation. In HCT 116, however, ERK1/2 phosphorylation is elevated relative to the isogenic derivative, but Rap1 activity is unchanged. K-RAS is uniquely oncogenic in the colonic epithelium, though the molecular aspects of its oncogenic contribution are not necessarily conserved across cell lines. We therefore conclude that the oncogenic contribution of K-RAS is a function of its multifaceted functionality and is highly context-dependent.

Both transcriptional subtype and signaling network analyses have proved useful in cancer genomics research. However, these two approaches are usually applied in isolation in existing studies. We reason that deciphering genomic alterations based on cancer transcriptional subtypes may help reveal subtype-specific driver networks and provide insights for the development of personalized therapeutic strategies. In this study, we defined transcriptional subtypes for colorectal cancer (CRC) and identified driver networks/pathways for each subtype. Applying consensus clustering to a patient cohort with 1173 samples identified three transcriptional subtypes, which were validated in an independent cohort with 485 samples. The three subtypes were characterized by different transcriptional programs related to normal adult colon, early colon embryonic development, and epithelial mesenchymal transition, respectively. They also showed statistically different clinical outcomes. For each subtype, we mapped somatic mutation and copy number variation data onto an integrated signaling network and identified subtype-specific driver networks using a random walk-based strategy. We found that genomic alterations in the Wnt signaling pathway were common among all three subtypes; however, unique combinations of pathway alterations including Wnt, VEGF and Notch drove distinct molecular and clinical phenotypes in different CRC subtypes. Our results provide a coherent and integrated picture of human CRC that links genomic alterations to molecular and clinical consequences, and which provides insights for the development of personalized therapeutic strategies for different CRC subtypes.

Lrig1 is an intestinal stem cell marker important for epithelial homeostasis. However, the position of the Lrig1 + population in the intestinal crypt has been debated, largely due to discrepant staining patterns using two Lrig1 antibodies. Here, we set out to decipher the differences between these Lrig1 antibodies to clarify their use for Lrig1-related studies. We confirmed the commercially available Lrig1-R&D antibody stained the bottom third of the colonic crypt, whereas an independently generated Lrig1-VU antibody recognized a subset of anti-Lrig1-R&D + cells. Biochemically, we found that anti-Lrig1-VU recognized a non-glycosylated form of Lrig1; in contrast, anti-Lrig1-R&D recognized both glycosylated and non-glycosylated forms of Lrig1. In addition, we generated a reporter mouse (Lrig1-Apple) as an independent readout of Lrig1 transcriptional activity. Flow cytometry of isolated colonic epithelial cells from Lrig1-Apple mice demonstrated anti-Lrig1-R&D recognized mostly RFP-hi cells, while anti-Lrig1-VU recognized cells that were largely RFP-mid. We conclude anti-Lrig1-R&D appears to recognize all Lrig1 + cells, while anti-Lrig1-VU recognizes a subpopulation of Lrig1 + cells.

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted miRNAs, we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild type KRAS exosomes. miR-10b was selectively increased in wild type exosomes while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild type recipient cells. These findings suggest extracellular miRNAs can function in target cells and uncover a potential...

The NIH Extracellular RNA Communication Program's initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell functio...

Composition and behavior of mutant KRAS exosomes 2 Abbreviations Amphiregulin (AREG), colorectal cancer (CRC), coefficient of variation (CV), epidermal growth factor receptor (EGFR), false discovery rate (FDR), multiple reaction monitoring (MRM), peptide to spectrum match (PSM), standard deviation (SD), standard error of the mean (SEM), transmission electron microscope (TEM), trifluoroethanol (TFE), voltage-dependent anion channel (VDAC)

instructed to select ≥1 diagnosis for each vignette. Results: 1) Total diagnostic agreement between all G was 2/20 vignettes, none among all F. IR percentage of agreement coefficient per case was >50% in only 7/20 vignettes (G) and 6/20 cases (F), <25% in 2/20 vignettes (G) and 3/20 vignettes (F). Average percentage of agreement coefficient among raters was G: 51%, range 22-100%, F: 46%, range 20-100%. Percentage agreement is considered inadequate when <70%. 2) To correct for possible agreement by chance, we calculated kappa coefficient (k), measure of pair-wise agreement. k was G: 0.47 (z = 21.3;p<0.0001), F: 0.42 indicating that the agreement was 47% and 42% better respectively than would be expected on basis of chance alone (k is considered as slight agreement between 0-0.2, fair 0.2-0.4, moderate 0.4-0.6, substantial 0.6-0.8, almost perfect 0.8-1). 3) We analyzed 2 separate subgroups: pain & constipation related disorders, IR percentage of agreement coefficient per case in constipation: 27-100%, mean 55% and 21-80%, mean 42% in pain subgroup. K value constipation: 0.3603; z = 26.6; p < 0.0001; k value pain: 0.31; z = 42.1762; p<0.0001. Conclusion: IR reliability of the Rome III criteria in pediatric gastroenterologists and fellows is moderate. Further validation of the Rome III criteria is necessary.