Over-Expression of Endothelin 3 Sensitizes Mice to Uv-Induced Melanoma Metastasis

Melanomagenesis is influenced by environmental and genetic factors. In normal cells, ultraviolet (UV) induced photoproducts are successfully repaired by the nucleotide excision repair (NER) pathway. Mice carrying mutations in the xeroderma pigmentosum (Xp) complementation group of genes (Xpa-Xpg) lack the NER pathway and are therefore highly sensitive to UV light; however, they do not develop melanoma after UV exposure. In humans, the Endothelin 3 signaling pathway has been linked to melanoma progression and its metastatic potential. Transgenic mice that over-express Edn3 under the control of the Keratin 5 promoter (K5-Edn3) and exhibit a hyperpigmentation phenotype, were crossed with Xp deficient mice. Because melanoma is highly metastatic and many primary malignancies spread via the lymphatic system, analyzing the lymph nodes may serve useful in assessing the possible spread of tumor cells to other tissues. This study aimed to determine whether the over-expression of Edn3 is sufficient to lead to melanoma metastasis to the lymph nodes. Mice were exposed to UV radiation and analyzed for the presence of skin lesions. Mice presenting skin lesions were sacrificed and the nearest lymph nodes were excised and examined for the presence of metastasis. Mice with melanoma skin lesions presented enlarged and hyperpigmented lymph nodes. Diagnosis of melanoma was established by immunostaining with melanocyte and melanoma cell markers, and while UV radiation caused the development of skin lesions in both K5-Edn3 transgenic and control mice, only those mice carrying the K5-Edn3 transgene were found to develop melanoma metastasis to the lymph nodes. These results indicate that over-expression of Edn3 is sufficient to lead to lymph node metastasis in mice exposed to at least one dose of UV radiation.

PROTEOMIC ANALYSIS OF WOLBACHIA SYMBIOSIS WITHIN THE DROSOPHILA OVARY

Wolbachia pipientis are bacterial endosymbionts carried by millions of invertebrate species, including ~40% of insect species and some filarial nematodes. In insects, basic Wolbachia research has potential applications in controlling vector borne disease. Conversely, Wolbachia of filarial nematodes are causative agents of neglected tropical diseases such as lymphatic filariasis and African river blindness. However, remarkably little is known about how Wolbachia interact with their hosts at the molecular level. Understanding this is important to inform the basis for symbiosis and help prevent human disease. I used a high-throughput proteomics approach to study how Drosophila host cells are modified by Wolbachia infection. This analysis identified 23 Drosophila proteins that significantly changed in amount as a result of Wolbachia infection. A subset of differentially abundant host proteins were consistent with Wolbachia-associated phenotypes reported previously. This study also provides the first ever discovery-based evidence for a Wolbachia-associated change in maternal germline histone loads, which has possible implications in Rescue of a common Wolbachia-induced reproductive manipulation known as Cytoplasmic Incompatibility.

Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Proteomic Analysis of Wolbachia Symbiosis within the Drosophila

Wolbachia pipientis are bacterial endosymbionts of arthropods and in some filarial nematodes. Wolbachia are of particular interest because nematodeWolbachia have been shown to cause the diseases African river blindness and Lymphatic Filariasis. Doxycycline can be used to eliminate nematode Wolbachia, however, more efficient treatments are needed. Ideally, we would like to repurpose another FDA approved drug that helps to shorten treatment duration. Vitamins are one of the best classes of FDA approved compounds, generally recognized as safe. Interestingly, prior work by Serbus and colleagues found that dietary yeast, which is highly enriched in vitamins, dramatically reducesWolbachia titer in Drosophila melanogaster ovarian tissue. Imaging data indicated that the Wolbachia nucleoids were disrupted in response to yeast. This raised the possibility that yeast cells contain a bio-reactive, anti-Wolbachiacompound. Our close examination of yeast nutritional information identified which vitamins are most highly enriched in yeast. We then administered several of these to D. melanogaster, and saw that two of these led to reduced ovarianWolbachia titers, analogous to yeast-fed flies. This was especially interesting, as both vitamins are critical for functioning of the same biochemical pathway. We used retested effect of one of these vitamins in oogenesis by performing a dilution series, and achieved positive correlation from this dilution series. This opens up the avenue for clarifying the mechanism of how vitamins suppressWolbachia titer, and for testing enhancement of Doxycycline, to hopefully provide faster, more affordable treatment for millions of patients.

Proteomic Analysis of Wolbachia Symbiosis within the Drosophila

Wolbachia pipientis are bacterial endosymbionts of arthropods and in some filarial nematodes. Wolbachia are of particular interest because nematodeWolbachia have been shown to cause the diseases African river blindness and Lymphatic Filariasis. Doxycycline can be used to eliminate nematode Wolbachia, however, more efficient treatments are needed. Ideally, we would like to repurpose another FDA approved drug that helps to shorten treatment duration. Vitamins are one of the best classes of FDA approved compounds, generally recognized as safe. Interestingly, prior work by Serbus and colleagues found that dietary yeast, which is highly enriched in vitamins, dramatically reducesWolbachia titer in Drosophila melanogaster ovarian tissue. Imaging data indicated that the Wolbachia nucleoids were disrupted in response to yeast. This raised the possibility that yeast cells contain a bio-reactive, anti-Wolbachiacompound. Our close examination of yeast nutritional information identified which vitamins are most highly enriched in yeast. We then administered several of these to D. melanogaster, and saw that two of these led to reduced ovarianWolbachia titers, analogous to yeast-fed flies. This was especially interesting, as both vitamins are critical for functioning of the same biochemical pathway. We used retested effect of one of these vitamins in oogenesis by performing a dilution series, and achieved positive correlation from this dilution series. This opens up the avenue for clarifying the mechanism of how vitamins suppressWolbachia titer, and for testing enhancement of Doxycycline, to hopefully provide faster, more affordable treatment for millions of patients.