<p> Every year in the US, over 200,000 men are diagnosed with prostate cancer (PCa) and annual mortality in the US exceeds 30,000. PCa is treated surgically, and post-surgical recurrent tumors treated through targeted inhibition of the androgen receptor (AR) and AR signaling through disruption of androgen synthesis. If tumors resist antiandrogen therapy, castration recurrent (CR) tumors are treated with second line therapies that currently involve more potent inhibition of AR. Since the advent of these newer therapies, alternative, AR-independent resistance mechanisms have begun to become more prevalent. Thus, there is a need to explore and discover novel pathways, drug targets, and mechanisms driving aggressive and atypical subtypes of PCa. </p><p> Non-coding RNAs (ncRNAs) are known to play critical roles in normal cell behavior, as well as various diseases including cancer. Small nucleolar RNAs (snoRNAs) are one class of ncRNAs that are primarily involved in guiding specific enzymatic modifications (e.g. 2&rsquo;O-methylation, pseudouridylation) of ribosomal RNAs (rRNAs) and thereby allowing fidelity of ribosome biogenesis. HBII-52 is an orphan C/D box snoRNA encoded in tandem repeat copies at chromosomal locus 15q11-13. HBII-52 has no predicted or reported rRNA targets, however, it has both predicted and validated messenger RNA (mRNA) targets, including the serotonin 2c receptor (5-HT2cR, encoded by the gene HTR2C). 5-HT2cR is a G-Protein Coupled Receptor (GPCR) predominantly expressed in brain, and it controls appetite and signaling via cognate ligand, serotonin (5-HT) binding. The pre-mRNA encoding 5-HT2cR is subject to complex alternative processing including alternative pre-mRNA splicing and Adenosine-to-Inosine (A-to-I) RNA editing. HBII-52 promotes processing to, highly active isoforms of 5-HT2cR, thus potentiating its signaling axis. A variety of inhibitors of 5-HT2cR exist including potent and selective inhibitors like SB242,084 that have already undergone preclinical evaluation for neurologic disorders. </p><p> Herein, we report that the expression levels of HBII-52 (MBII-52 in mouse) and 5-HT2cR are deregulated in PCa including both mouse and human preclinical models, and human clinical specimens. Furthermore, mechanistic studies of the HBII-52/5-HT2cR pathway in PCa cells indicated this pathway drives transition to an aggressive and invasive phenotype, specifically by a highly active, less edited isoform of 5-HT2cR. Finally, we evaluated the feasibility and efficacy of targeting 5-HT2cR using small molecules in PCa and arrived at a lead drug candidate, SB242,084. In conclusion, active edit-isoforms of 5-HT2cR promote aggressiveness and invasiveness of PCa cells. One potential mode of activation includes upregulation of HBII-52. HBII-52/5-HT2cR-positive cancer cells can be effectively targeted through selective inhibition of 5-HT2cR with small molecules.</p>

Thesis (Ph. D.)--University of Toronto, 2008. / Includes bibliographical references.

<p> Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is caused by the loss of laminin-211 and laminin-221 heterotrimers which are most abundant in skeletal and cardiac muscle basal lamina; mutations in the LAMA2 gene cause the loss of these laminin isoforms. This absence of laminin-211/221 in MDC1A reduces the capacity for myofiber adhesion, loss of sarcolemmal integrity and subsequently the ability of the skeletal muscle syncytium to generate force in a coordinated and efficient manner. Patients experience progressive muscle wasting which confines them to a wheelchair at an early age and respiratory failure that leads to their untimely death. Currently, there is no effective treatment or cure for this devastating disease. Previous studies have shown that laminin-111, an embryonic form of laminin, delivered before disease onset can reduce muscle pathology and improve viability in the dyW-/- mouse model of MDC1A. These studies suggested that laminin-111 may act to strengthen and reinforce the sarcolemma and provide a protective niche for muscle repair. Since most patients are diagnosed with MDC1A after disease onset, we determined if laminin-111 could be beneficial after disease onset. Our studies suggest dyW-/- mice treated with laminin-111 after disease onset show improvement in muscle function and histology. Results from this study along with an understanding of laminin-111 pharmacokinetics will help pave the way in developing this protein as an exciting potential therapeutic for MDC1A patients. Duchenne Muscular Dystrophy (DMD) is the most common X-linked disease affecting 1 in 3,300 live male births. Patients with DMD suffer from severe, progressive muscle wasting and weakness with clinical symptoms first detected between 2 to 5 years of age; as the disease progresses patients are confined to a wheelchair in their teens and die in their early 20s mainly due to cardiopulmonary complications. DMD is caused by the loss of the sarcolemmal protein dystrophin (427kDa) due to mutations in the dystophin gene. When present, dystrophin acts as a scaffold linking the cell cytoskeleton to the extracellular matrix. This loss of dystrophin in DMD results in patients experiencing greater susceptibility to muscle damage via reduced structural and functional integrity of their muscle. One potential therapeutic avenue that needs to be explored involves increasing the levels of the ?7?1 integrin in order to compensate for the loss of dystrophin. To test this hypothesis, a muscle cell-based assay was developed in order to report ?7 integrin promoter activity with the intent of identifying molecules that promote ?7 integrin expression. Laminin-111 was identified as an enhancer of ?7 integrin expression. Theoretically, the identification of ?7 integrin enhancing compounds that help boost ?7?1 integrin expression as part of drug-based therapies may lead to a novel therapeutic approach for the treatment of this disease. Systemic laminin-111 treatment significantly reduces myofiber degeneration in both forms of MDC1A and DMD muscular dystrophy. This dissertation reinforces the potential of laminin-111 as a systemic protein therapy, capable of restoring sarcolemmal integrity thus reducing muscle disease progression. The importance of ?7 integrin in skeletal and cardiac muscle was highlighted here through the generation of the ?7-/-:: laminin-?2-/- double knockout mouse model. This mouse has never been studied before and could prove to be another important mouse model needed to explore therapeutic avenues for muscular dystrophy.</p>

<p> Extracellular adenosine signaling via the adenosine receptor 2B (AR_2B) is a potent profibrotic and proinflammatory molecule that has been shown to contribute to tissue remodeling and excessive matrix protein production under chronic conditions in kidney disease. Investigation of the adenosine pathway on interstitial renal fibroblasts is lacking even though fibroblasts are a major source of ECM production and contribute to the pathogenesis of renal failure in late stage kidney disease. Analysis of mRNA from the interstitial renal fibroblast cell line NRK-49F revealed these cells express AR₁ and AR_2B. Utilizing highly selective small molecule agonists and antagonists specific to each individual adenosine receptor, it was determined by mobilization of intracellular cAMP, both receptors were functional. AR_2B was observed to be the dominant receptor under high &mu;M concentrations of agonism leading to increased pro-inflammatory and profibrotic mediators. Specifically, agonism of AR_2B lead to increased levels of &alpha;SMA and other inflammatory and fibrotic mediators, indicating a transdifferentiation of fibroblasts to a more activated myofibroblasts stage under strong and sustained adenosine signaling. The key fibrotic mediator TGF-&beta;1 was observed to be increased with adenosine signaling while antagonism of AR_2B suppressed TGF-&beta;1 mediated increases in &alpha;SMA and fibronectin, suggesting complementary signaling pathways with significant crosstalk. It was further observed that AR_2B receptor antagonism suppressed TGF-&beta; receptor mediated SMAD binding element associated transcriptional activity the suppression of TGF-&beta; receptor signaling and transcriptional activity was associated with decreased SMAD4, a critical protein necessary for proper formation of the SMAD complex transcription factor. Taken together these data highlight the impact of adenosine signaling on proinflammatory and profibrotic endpoints in renal fibroblasts and describe for the first time direct inhibition of TGF-&beta; receptor signaling by antagonism of the AR_2B receptor.</p><p>

No description available.