published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Chinese Medicine / Master / Master of Philosophy

Tamoxifen is the most widely used endocrine therapy for both early and advanced estrogen receptor (ER) positive breast cancer patients. About half of the patients that initially respond to the antiestrogen become estrogen-independent and ultimately develop resistance to the treatment. The precise molecular mechanisms of tamoxifen resistance remain poorly understood. Dysregulation of microRNAs (miRNAs) has been frequently reported in breast cancer and linked to cancer development, progression and therapeutic response. To gain a more comprehensive picture of the miRNA regulatory network for modulating tamoxifen responsiveness, we examined global expression profiles of more than 600 miRNAs in a matched pair of tamoxifen-sensitive ZR75 and tamoxifen-resistant AK47 breast cancer cell lines using TaqMan Low Density Array (Applied Biosystems). Under 4-hydroxytamoxifen treatment, 102 miRNAs displayed differential responses between the sensitive cells and the resistant cells. At basal levels, upregulation of 32 miRNAs and downregulation of 75 miRNAs were observed in the resistant cells as compared to the sensitive cells. Among the 9 miRNAs of significant differential expression selected for validation, expression profiles of the 7 miRNAs could be reproduced. Of these, 4-hydroxytamoxifen treatment greatly increased miR-449a/b expression in sensitive ZR75 cells, whereas miR-449a/b expression was significantly reduced in resistant AK47 cells at basal levels, which was further confirmed in a panel of tamoxifen-resistant breast cancer cell lines. Such downregulation of miR-449a/b in the resistant cells was partially attributed to DNA methylation-mediated repression of miR-449a/b. Notably, miR-449a/b expression exhibited a significant positive correlation with ER-α status (miR-449a: P=0.006, miR-449b: P=0.013) and progesterone receptor (PR) status (miR-449a: P=0.010, miR-449b: P=0.021), and a prominent inverse association with tumor grade in 61 breast cancer tissues (miR-449a: P=0.001; miR-449b: P=0.009). Also, breast cancer patients with high miR-449a/b expression tended to have increased disease-free survival (miR-449a: P=0.019; miR-449b: P=0.117). To further support the tumor suppressor function of miR-449, stable miR-449b overexpression in the resistant cells reduced cell proliferation. More intriguingly, restoring miR-449b expression increased sensitivity to 4-hydroxytamoxifen-induced apoptosis via suppression of AKT activity without restoring ER-α expression. In contrast, miR-449a/b knockdown reduced ER-α and PR expression, but enhanced phosphorylation of AKT, extracellular signal-regulated kinase- 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and also ER-α at serine 167 and serine 118 residues. Furthermore, we demonstrated c-Myc is a target gene of miR-449 as confirmed by bioinformatics and experimental analyses. Computational algorithms predicted a highly conserved miR-449a/b binding site within C-MYC 3’untranslated region (3’UTR). Compared to the parental sensitive cells, c-Myc was overexpressed in the resistant cells. Forced expression of miR-449b suppressed c-Myc protein level. To further support the notion that c-Myc is a direct target of miR-449, interactions between miR-449b and C-MYC 3’UTR were confirmed by co-expression of miR-449b and c-Myc expression constructs and luciferase reporter assay. Taken together, our data strongly suggest the critical role of miR-449 in modulating altering response to tamoxifen via targeting c-Myc. Suppression of miR-449 repressed genomic ER action and concomitantly activated non-genomic ER pathways. These findings may provide insights to improve breast cancer management and open a wide avenue for therapeutic interventions for overcoming tamoxifen resistance. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Epstein-Barr virus (EBV) is one of the most successful human pathogens in the world and establishes a lifelong persistent infection in 95% of adult population worldwide. It is associated with a number of malignancies including Burkett’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma(NPC) and gastric carcinoma. EBV was the first virus reported to produce microRNAs (miRNAs) and it encodes 44 mature miRNAs from 2 viral transcripts, BART and BHRF1. The BART transcript is abundantly expressed in all latently infected cells, particularly in epithelial cells. The BART miRNAs (miR-BARTs) were shown to be involved in apoptosis inhibition, immune evasion, metastasis, viral and cellular transcripts regulation. The high expression profile and the diverse functions of miR-BARTs suggest that they may play a critical role in the development of EBV-associated NPC. In order to understand the importance of miR-BARTs in NPC development, in this thesis, I conducted a study on the miR-BARTs function in nasopharyngeal carcinogenesis. In the first part, I characterized the cellular target and function of an abundantly expressed miR-BART in NPC. In the second part, I established a novel recombinant EBV construction system for genetic studies of miR-BARTs in nasopharyngeal epithelial (NP) cells. In the first part of my study, I characterized the cellular target and function of miR-BART3* in NPC. As predicted by bioinformatics, tumor suppressor protein DICE1 was a cellular target of miR-BART3*. The specific targeting between miR-BART3* and DICE1 3’UTR was validated by luciferase assays and the downregulation of both endogenous DICE1 protein and mRNA was observed in EBV+epithelial cells and miR-BART3* expressing cells. In addition, restoration of DICE1 protein expression by inhibition of miR-BART3* was also demonstrated in EBV+epithelial cells. Moreover, miR-BART3* was shown to promote cell proliferation via suppression of DICE1. Analysis of22 human nasopharyngeal(NP)biopsy samples demonstrated the inverse correlation between miR-BART3* and DICE1 expression. Taken together, miR-BART3* downregulates the tumor suppressor DICE1 protein to promote cell proliferation and transformation in NPC. Besides the candidate approach, genetic studies can provide a systematic view of the functions of all miR-BARTsand shed light on the importance of miR-BARTs in NPC under a more physiological condition. At present, bacterial artificial chromosome (BAC) technology is commonly used for recombinant EBV construction. However, the intrinsic disadvantages of BAC prevent its use in NP epithelial cells. Therefore in the second part of my study, I established a novel CRISPR/Cas9-mediated recombinant EBV construction system and constructed a miR-BART deleted recombinant EBV. The CRISPR/Cas9 system was demonstrated to be effective in EBV genome editing and Akata cells were infected by the recovered recombinant mutant virus. Infected Akata cells served as the source for NP cell infection through co-culture. The new CRISPR/Cas9 system have many advantages over the conventional EBV BAC method. My work reported in this thesis not only illustrated the importance of miR-BARTs in NPC, but also provide a new technology platform for further study of miR-BARTs in NP epithelial cells. (An / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Melanoma is the most deadly form of skin cancers, with an incidence increasing more rapidly than any other malignant cancer in the past 40 years. Metastatic melanoma is resistant to conventional treatments, such as chemotherapy and radiation therapy. Our lab has previously demonstrated that Mcl-1 is a key contributor in protecting melanoma from therapy-induced cell death. RNAi therapeutics was employed as a novel way to silence the anti-apoptotic protein by using Mcl-1 mRNA sequence-specific siRNAs in vitro. In our hands, passive non-targeted delivery of RNAi therapy into melanoma tumours has been shown to be neither effective, nor selective in vitro and in vivo. Consequently, in this study, siRNA was linked to a delivery system which expressed a ligand specifically targeting melanoma cells. Previously shown, melanoma overexpresses the cell surface ganglioside GD3, thus it is my belief that the anti-GD3 R24 monoclonal antibody can function as a targeting molecule. The antibody was linked to coated cationic liposomes (CCLs) carrying siRNA molecules. Our first step was to confirm R24 ligation to CCLs. Untargeted CCLs showed insignificant values of antibody, whereas antibody-conjugated CCLs presented approximately 30 antibodies per liposome. I also confirmed that siRNA was internalized within CCLs using spectrometry, with an encapsulation efficiency of approximately 80%. Since liposomes need to be small to be effective in vitro and in vivo, CCLs were confirmed to be less than 100nm in diameter. In vitro studies using fluorescent microscopy demonstrated greater binding to melanoma cells with antibody-conjugated CCLs as compared to untargeted CCLs. In vivo experiments showed specific localization of targeted CCLs to induced subcutaneous mouse xenograft tumours. Western blotting demonstrated greater Mcl-1 knockdown using GD3-targeted CCLs. Taken together, these results suggest that anti-GD3 antibodies can serve as targeting molecules to deliver siRNA to melanoma cells and furthermore, GD3-targeted CCLs can promote siRNA-mediated gene silencing. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-09-02 10:29:37.944

Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare due to the devastating diseases these viruses inflict on humans, animals and plants. Rapid viral RNA evolution is frequently due to RNA recombination, which can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. We have demonstrated that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. Our results support the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses. To test if host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes ~80% of yeast genes, in RNA recombination studies based on a small viral replicon RNA derived from TBSV. The genome-wide screen led to the identification of five host genes, whose absence resulted in rapid generation of novel viral RNA recombinants. Thus, these genes normally suppress viral RNA recombination, but in their absence hosts become viral recombination hotbeds. Four of the five recombination suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA recombination. Overall, our results demonstrate for the first time that a set of host genes have major effect on RNA virus recombination and evolution. Replication of the non-segmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. We have demonstrated that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Our findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

T-cell acute lymphoblastic leukemia (T-ALL) is a childhood cancer created through genetic alterations; most commonly upregulation of TALI and LMOI oncoproteins. T-ALL is treated with radiation and chemotherapy, but malignant T-cells are resistant to apoptotic stimulation. To study this disorder, AKR-DP-603 cells were transduced to express both oncoproteins. Western blots verified protein expression and each population was treated with etoposide. Caspase-3 and Annexin-V/FITC apoptosis assays were performed following treatment. When the response of control cells was compared to engineered cells, no difference was observed from the Annexin-V/FITC assay, and only LM01 cells showed a difference in the caspase-3 assay. Furthermore, cells were transfected with siRNA to TALI and LM01 and the apoptotic response was re-tested. Complete silencing was verified by Western and apoptotic activity varied in the TALI population for both assays. These differences might indicate that cells resisted etoposide induction and following silencing were sensitized apoptotic induction. / Department of Biology

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb 15, 2008). Includes bibliographical references.

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 98-102.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.