Adaptor proteins play a vital role in the propagation of cellular signals. Although they lack endogenous catalytic activity, they contain a variety of protein binding modules, which enable them to promote specific and efficient interactions with their binding partners. They form integrative platforms for a variety of molecules (e.g. lipids, tyrosine kinases, cytoskeletal and signaling proteins), and thereby link and coordinate key functions such as cell growth, motility and shape determination. Our laboratory has recently cloned a novel, 130 kDa adaptor protein, named XB130, as a structural homolog of actin-filament-associated-protein (AFAP-110), a stress fiber-binding Src substrate. However, the molecular interactions and functions of this novel adaptor remained to be elucidated. To characterize the function of XB130 we asked two general questions: (1) Is XB130 involved in the signal transduction pathways of tyrosine kinases? And (2) Is XB130 capable of regulating the cytoskeleton and/or is it regulated by the cytoskeleton? To address these questions first we investigated the tissue distribution of XB130 and discovered that it is abundantly expressed in thyroid. Therefore we asked whether it is a target of the thyroid-specific tyrosine kinase, RET/PTC, a genetically rearranged, constitutively active enzyme that plays a pathogenic role in papillary thyroid cancer. We found that XB130 is a RET/PTC substrate that couples RET/PTC signaling to phosphatidylinositol 3-kinase (PI3K) activation through its phosphorylation dependent interaction with the regulatory subunit p85 of PI3K. XB130 plays an important role in PI3K signaling, as downregulation of XB130 in TPC1 papillary thyroid cancer cells, harboring the RET/PTC1 kinase, strongly reduced Akt activity and concomitantly inhibited cell cycle progression and survival in suspension. In the second part we demonstrate that XB130 is a novel Rac- and cytoskeleton-regulated protein that exhibits high affinity to lamellipodial (branched) F-actin and impacts motility and invasiveness of tumor cells. In conclusion, my work characterized a novel adaptor protein and assigned two well-defined pathophysiological functions to it in the context of thyroid cancer cells.

The integrin family of cell surface receptors is evolutionary conserved and found in all multicellular animals. In humans 8-alpha and 18-beta integrins are non-covalently associated into 24 dimers. Integrins mediate cell-extracellular matrix and cell-cell interactions and participate in cell signalling. This ideally places integrins to regulate vital processes such as cell adhesion, migration, differentiation and cytoskeleton dynamics. Integrins also play a fundamental role in regulating cell survival and anoikis. In this thesis molecular mechanisms employed by integrins to induce signal transduction, independently or through crosstalk with other receptors, were characterised. Rictor-mTOR (mTORC2) was required for Akt Ser473 phosphorylation in response to β1 integrin-mediated adhesion as well as EGF-, PDGF- or LPA-stimulation of MCF7 cells. ILK and PAK were dispensable for Akt Ser473 phosphorylation upon β1 integrin-engagement or EGF-stimulation. PAK was needed when this phosphorylation was induced by PDGF or LPA. β1 integrin-promoted cell survival during serum starvation conditions was mTORC2 dependent, indicating the importance of Akt Ser473 phosphorylation. mTORC2 was also required for Akt Ser473 phosphorylation induced upon heparanase treatment of cells. Heparanase preferred PI3K catalytic subunit p110α for the upstream lipid phosphorylation required for Akt activation. Interaction between this subunit and Ras was needed for optimal Akt phosphorylation upon heparanase exposure. Cell adhesion strongly promoted heparanase signalling, which was more efficient in β1 integrin-expressing fibroblasts compared to cells lacking this subunit. The cooperative signalling between integrins and heparanase involved FAK and PYK2 since simultaneous silencing of these kinases suppressed heparanase-triggered Akt activation. Furthermore, the resistance of cells to apoptosis induced by H2O2 or serum starvation was promoted by heparanase.  Integrin stimulation by adhesion or cyclic stretching showed divergent downstream signalling responses. Cell attachment on integrin-specific ligands lead to robust phosphorylation of several intracellular integrin-effectors, e.g. p130CAS, FAK, Akt and ERK 1/2. However, mechanical cell stretching only triggered prominent phosphorylation of ERK 1/2. Signalling induced at early stages of integrin-mediated cell adhesion occurred independently of intracellular contraction. Reactive oxygen species (ROS) generated during adhesion and cell stretching influenced integrin signalling. Inhibition of mitochondrial ROS production blocked adhesion-induced Akt phosphorylation. In contrast, stretch-induced ERK 1/2 phosphorylation was elevated when extracellular ROS was scavenged. These results indicate that the two types of integrin stimuli generate signals by different mechanisms.

No description available.

No description available.

The PIK3R1 gene encodes three proteins - p85$\alpha$, p50$\alpha$ and p55$\alpha$ - that are regulatory subunits of Class IA phosphoinositide 3-kinases (PI3Ks). These regulatory subunits heterodimerise with one of three catalytic subunit isoforms, namely p110$\alpha$, p110$\beta$, or p110$\delta$. Class IA PI3Ks are critical enzymes involved in fundamental metabolic and mitogenic signalling pathways. This thesis describes the delineation of biochemical and molecular mechanisms whereby PIK3R1 mutations cause diverse disease phenotypes observed in SHORT syndrome (defined by Short stature, Hyperextensibility, Ocular depression, Rieger anomaly and Teething delay), the primary immunodeficiency Activated PI3K-$\delta$ Syndrome 2 (APDS2), and cancer. Initial studies of purified wildtype or mutant PI3K complexes, utilising a modified PI3K fluorescence polarisation lipid kinase assay, established that SHORT syndrome-associated p85$\alpha$ mutations impaired phosphotyrosine peptide-stimulated PI3K activity when heterodimerised with either of the Class IA catalytic subunit isoforms. Two cancer-associated mutations assessed using the same assay demonstrated differential effects on PI3K function, causing either basal activation or impaired phosphotyrosine peptide-stimulated PI3K activity. To examine the effect of SHORT syndrome-associated p85$\alpha$ mutations in insulin-responsive cell types, 3T3-L1 preadipocyte models with conditional overexpression of p85$\alpha$ Y657X or p85$\alpha$ R649W were generated. Doxycycline-induced overexpression of mutant p85$\alpha$ attenuated insulin-stimulated Akt phosphorylation due to reduced insulin-stimulated association of p85$\alpha$/p110$\alpha$ heterodimers with either IRS1 or IRS2. This in turn resulted in impaired downstream signalling as indicated by low adipogenic efficiency. Cells and tissues isolated from Pik3r1$^{WT/Y657X}$ knock-in mice also demonstrated decreased insulin-stimulated Akt phosphorylation. Observations from a system with endogenous expression of mutant p85$\alpha$ Y657X supported the results obtained in the 3T3-L1 p85$\alpha$ overexpression models. The final part of this thesis focussed on a PIK3R1 exon skipping mutant (p85$\alpha$ $\Delta$Ex11) that confers PI3K activation in lymphocytes and causes APDS2. APDS2 patients have an immune-restricted phenotype, even though the mutation occurs within the ubiquitously expressed PIK3R1. To investigate this phenomenon, the doxycycline-inducible system was used to model overexpression of p85$\alpha$ $\Delta$Ex11, as well as an activating p110$\alpha$ H1047R mutation associated with cancer, in 3T3-L1 preadipocytes. Surprisingly, given that APDS2 is not normally associated with metabolic or growth problems, high overexpression of p85$\alpha$ $\Delta$Ex11 severely attenuated insulin-stimulated Akt phosphorylation and adipocyte differentiation. There was also reduced insulin-stimulated recruitment of p110$\alpha$ to either IRS1 or IRS2, and impaired heterodimerisation of p85$\alpha$ $\Delta$Ex11 with p110$\alpha$. Collectively, the data presented in this thesis contributes to the developing knowledge of PIK3R1-related diseases. In particular, these studies provided novel insights into the biochemical and molecular mechanisms of SHORT syndrome-associated p85$\alpha$ mutations. Additionally, these data delivered further understanding of potential mechanisms underlying the immune-specific phenotype of APDS2 caused by PIK3R1 mutations.

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

7Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by accumulation and deposition of extracellular beta-amyloid peptides (Aβ) and intra-neuronal hyperphosphorylated tau in the brain. The insulin signalling pathway begins upstream at the insulin receptor (IR), where the intracellular insulin receptor substrate 1 (IRS1) is phosphorylated, thus propagating the signal downstream to the PI3K/Akt signalling pathway, which affects both the glycogen synthase kinase 3 beta (GSK3β), which is a tau kinase, and mTOR, which is a critical part of the mTORC1 and mTORC2 complexes that not only mediate a wide range of cell functions, but also feed back upstream to regulate Akt. Increasing evidence builds a strong case for the role of soluble Aβ oligomers (AβOs) in the impairment of insulin signalling in AD. Our in vitro studies with neuroblastoma 2a (N2a) cells stably transfected with human APP695 gene (N2a-APP), which secrete excess Aβ, show that the phosphorylation and expression of several but not all critical signalling proteins along the insulin signalling pathway are dysregulated in the cells in comparison to the parental N2a cells. N2a-APP cells were also found to be phenotypically insulin resistant. Subsequently, N2a-APP cells were treated with the Aβ binding peptide (ABP), which binds Aβ oligomers. The ABP treatment was observed to enhance insulin signalling response compared to untreated controls. The results suggest that Aβ may be responsible for inducing the insulin resistant phenotype in N2a-APP cells, and that the removal of Aβ oligomers is a potential treatment consideration for dysfunctional insulin signalling involved in Alzheimer’s disease.

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate similar to 30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4(+)/CD8(+) cells, we hypothesized that Program Death Ligand-1 (PDL1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3K alpha (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (similar to 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kappa B expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) similar to 30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI > 90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

No description available.

No description available.