v-Rel is the acutely oncogenic member of the NF-[kappa]B family of transcription factors and transforms cells through the altered regulation of pathways normally controlled by cellular NF-[kappa]B. Initial studies revealed that expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAP kinases. This induction is critical for the v-Rel transformed phenotype, as suppression of MAPK activity with chemical inhibitors or siRNA severely limited colony formation of v-Rel transformed cell lines of hematopoietic origin. However, signaling must be maintained within a certain range in these cells, as strong additional activation of either pathway through expression of constitutively active MKK mutants also attenuated the transformed phenotype. Studies in primary spleen cells revealed that MAPK signaling is also required for the early stages of v-Rel-mediated transformation. However, constitutive MAPK activity further enhanced the transformation efficiency of v-Rel in primary cells. These studies, as well as analogous experiments in DT40 cells, indicate distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation. The proto-oncoprotein, c-Rel, only weakly activates ERK and JNK signaling compared to v-Rel. Importantly, elevated MAPK activity enhanced transformation by c-Rel, indicating that the ability of v-Rel to induce MAPK signaling is a major contributor to its oncogenic potential. Taken together, this work demonstrates an important role for ERK and JNK activity in transformation by v-Rel. Additional studies examined mechanisms through which MAPK activity is regulated in v-Rel transformed cells. Feedback regulation of the ERK activator, MKK1, at T292 was shown to limit ERK activation in v-Rel transformed cells, preventing the detrimental effects of constitutive activity. This result is the first indication that this regulation may have a role in the maintenance of transformation. Further, several v-Rel induced cytokines were identified that activate ERK and JNK signaling in v-Rel transformed cells, revealing one means by which v-Rel-dependent transcriptional changes lead to MAPK activation. These studies demonstrate the integration of multiple mechanisms in achieving the optimal levels of MAPK activity that are essential for v-Rel-mediated transformation. / text

The small GTPases function as molecular switches to control diverse signaling cascades. The mammalian Rit and Rin, along with Drosophila Ric, comprise an evolutionarily conserved subfamily of the Ras-related GTPases. Previous studies using cultured cell models suggested that Rit was involved in the control of cell proliferation, transformation, neuronal differentiation, morphogenesis, and cell survival, but the principal physiological function of Rit remained uncharacterized. To address this outstanding question, we employed a genetic approach, engineering a Rit knockout mouse. Using this animal model, we demonstrate a central role of Rit in governing cell survival in a p38-dependent fashion. Primary mouse embryonic fibroblasts (MEFs) derived from Rit-/- mice display increased apoptosis and selective disruption of MAPK signaling following oxidative stress. These deficits include a reduction in ROS-mediated stimulation of a novel p38-MK2-HSP27 signaling cascade, which appears to act upstream of the mTORC2 complex to control Akt-dependent cell survival. In the adult brain, proliferation of stem cells within the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), provide a lifelong supply of new neurons. Adult neurogenesis appears critical for learning and memory and is altered in animal models of brain injury and neurological diseases. Thus, a greater understanding of the regulation of adult neurogenesis will provide insight into its myriad physiological roles but also to the development of therapeutic strategies for the treatment of injury and the progression of brain diseases. Here we find that Rit plays a central role in governing the survival of hippocampal neurons in response to oxidative stress. Importantly, using a controlled cortical impact model of traumatic brain injury (TBI), we show that Rit acts to protect newborn immature neurons within the SGZ of the DG from apoptosis following TBI. Finally, studies indicate that Rit plays a significant role in directing IGF-1 signaling, a key neurotrophin known to promote neurogenesis and to protect neurons against apoptotic stress. Together, these studies establish Rit as a critical regulator of a p38 MAPKdependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress, including the survival of newborn hippocampal neurons in the traumatically injured brain.

Pain from bone metastases is multifaceted with clinical descriptors including ongoing pain, hypersensitivity to external stimuli and intermittent episodes of breakthrough pain characterized as a sudden and abrupt onset of severe pain on a background of well-controlled pain. Moreover, cancer-induced bone pain remains inadequately managed due to a myriad of side effects associated with the current pain relieving regimens, which primarily rely on administration of opiates. Despite advances made in cancer therapeutics, these patients experience an inferior quality of life with incapacitating pain with limited daily activities. Development of long-term novel, non-opiate mechanism-based therapeutics with limited side effects is considered beneficial in elevating the patients' quality of life. First part of this dissertation encompasses the role of p38 MAPK in a mouse model of cancer-induced bone pain in which breast cancer cells were injected and sealed into the femur. Our data demonstrated that both acute and prolonged inhibition of p38 MAPK blocked cancer-induced spontaneous pain but had no effect on the evoked pain indicating important differences in mechanisms mediating ongoing pain as opposed to evoked pain. Undermanaged control of breakthrough pain is attributed to poor understanding of underlying mechanisms and how they may differ from ongoing pain due, in part, to lack of a pre-clinical model in which these mechanisms can be studied. We have established a rat model of cancer-induced bone pain to examine ongoing pain and pain relief using conditioned place preference paradigm as well as breakthrough pain using palpation-induced conditioned place aversion. We have shown that while peripheral afferent input from the tumor-bearing tibia mediates cancer-induced ongoing pain and initiation of breakthrough pain, it does not contribute to the maintenance of breakthrough pain. These data suggest that molecular targets mediating these two mechanisms may be different. This hypothesis was confirmed by our findings in this model that acute blockade of interleukin-6 blocked movement-evoked breakthrough pain in tumor-bearing rats, but failed to block tumor-induced ongoing pain. Hence, we provide a platform to manipulate treatments that can be given alone or in combination with opiates in such a way that patients receive adequate control of breakthrough pain.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

<p>The gene BRAF is mutated to remain aberrantly activated in a large number of human malignancies, most prominently in melanoma. The most common mutation in BRAF is a missense mutation that substitutes glutamic acid for valine at codon 600 (V600E) that leads to constitutive activation of this kinase. In this active state, BRAFV600E phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate the ERK1 and ERK2 kinases of the MAPK pathway to promote tumorigenesis. Targeting this pathway is a well-validated strategy to treat BRAF-mutant cancer. Inhibitors of both BRAFV600E and the MEK1/2 kinases are used to treat BRAF-mutant melanoma and are being evaluated in other cancers as well. However, the duration of response to these targeted therapies is limited by innate and acquired resistance, which is often mediated through reactivation of the MAPK pathway. Thus, new targeted therapies to inhibit MAPK signaling in BRAF-mutant malignancies are required. To this end, MEK1/2 kinases require copper (Cu) for enzymatic activity and signaling. We therefore tested whether the dependency of these validated targets on Cu could be leveraged for the treatment of BRAF-mutant cancer.</p><p>We report that genetic reduction of Cu import through disruption of the gene encoding the high affinity Cu transporter CTR1 or pharmacological chelation of Cu with the drug tetrathiomolybdate (TTM) suppressed MAPK signaling in both in vitro and in vivo models of BRAF-mutant tumorigenesis. This reduction in MAPK signaling correlated with a reduced potential for tumorigenic growth and an increase in survival of tumor-bearing mice. Finally, TTM reduced the transformed growth of a number of human melanoma cell lines engineered to be resistant to current MAPK pathway inhibitors. As such, Cu chelation holds promise as a novel treatment for BRAF-mutant cancers and may find value in targeting resistance to current MAPK pathway inhibitors.</p> / Dissertation

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.