Cigarette smoke (CS) exposure is the leading cause of preventable death in the United States contributing to over 480,000 deaths a year with over 300 billion dollars in CS related costs spent per year. While the dangers of CS exposure have been studied and characterized for decades being largely attributed to reactive oxygen species and oxidative stress, increasing evidence suggests that reactive aldehydes in CS, specifically the α,β-unsaturated aldehyde acrolein, are responsible for many of the negative pathologies associated CS exposure. Previous work has shown that acrolein can bind directly to a number of cellular proteins containing redox sensitive cysteine residues. The non-receptor tyrosine kinase Src contains nine cysteine residues and is known to be activated in response to CS and oxidative stress. Despite being the first characterized and one of the most widely studied oncogenes, the exact mechanism for Src activation remains unclear. In the current studies we examined the effects of acrolein on Src activation and the resulting outcomes on the lung epithelium in an effort to better understand how reactive electrophiles in CS contribute to the development of lung disease. To determine the effects of acrolein on Src activation, we first exposed NCI-H292 cells to acrolein and measured activity by western blot. We observed an increase in Src activity detected by an increase in Src phosphorylation at Y416 and an increase in phosphorylation of Src target proteins Caveolin1 and p120. Interestingly the increase in activation occurred without dephosphorylation of the inhibitory phosphorylated tyrosine Y527. Using biochemical-labeling strategies we identified Src as a direct target of acrolein adduction in vitro and in vivo, and we used mass spectrometry to confirm acrolein adduction to cysteine residues C245, C277 and C487, all which have been implicated in a redox dependent Src activation mechanism. Furthermore, increased Src activity following acrolein exposure was confirmed using an in vitro kinase activity assay and recombinant Src in a cell free system. To study the effects of acute acrolein exposure on lung epithelial function we exposed cultured mouse tracheal epithelial cells (MTECs) to acrolein and show impaired epithelial barrier function, measured by a decrease in trans epithelial resistance (TER) and increased epithelial permeability to FITC-dextran, which could be prevented using the Src inhibitor PP2. Src inhibition also attenuated acrolein-induced loss of E-cadherin and ZO-1. Acute exposure of C57BL/6 mice to acrolein (5 ppm for 4 hrs) led to increased epithelial permeability, measured by enhanced leakage of i.v. injected FITC-dextran into the airspaces, and induction of HO-1 in the lung while chronic acrolein exposure resulted features of epithelial to mesenchymal transition including a reduction of E-cadherin, increased vimentin, increased expression of MMP9 and increased collagen deposition. Chronic acrolein exposure in vitro resulted in a reduction of E-cadherin that could be prevented using the Src inhibitor AZD0530. Together our studies demonstrate that Src is a direct target for acrolein and plays an important role in epithelial alterations due to acrolein exposure. This work provides further insight into a potential mechanism involved in the development of cigarette smoke related disease and could provide a potential target for novel therapeutics.

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

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

Reproduction de : Thèse de doctorat : Pharmacie. Chimie thérapeutique : Nantes : 2008. / Bibliogr.

Approximately 30% of human breast cancers demonstrate overexpression of the receptor tyrosine kinase ErbB2/HER2/Neu, with these cases correlating with recurrence and poor prognosis. While therapy targeting ErbB2 has met with some success, particularly in early-stage breast cancers, transformation and progression towards a later-stage metastatic phenotype is likely sustained by aberrant signaling from additional players, for instance that downstream of integrins. In fact, treatment with integrin-blocking antibodies and ß1-integrin ablation leads to reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo. Moreover, ErbB2 has recently been found to interact with the ß4-integrin subunit to promote tumour formation and progression, as deletion of the ß4-integrin signaling domain led to suppression of mammary tumour onset and invasive growth, coupled with decreases in ErbB2-dependent signaling. ErbB2 may interact with integrin subunits by direct binding or via the intracellular kinases Src and focal adhesion kinase (FAK), both known to be activated downstream of ErbB2 and integrins and having well-established roles in cell adhesion, migration, and invasion. Using an inducible model of ErbB2 activation, we have demonstrated that controlled ErbB2 activation in human mammary epithelial cells leads to phosphorylation of Src Tyr215 and FAK Tyr861, consistent with a recently published clinical study examining phosphorylated forms of Src and FAK in ErbB2-positive human breast tumour samples. We have also confirmed that ErbB2 activation increases the capacity of cells for survival: Normally, MCF10A human mammary epithelial cells cultured in three-dimensional, laminin-rich extracellular matrix gel form mammary acini-like spheroids with hollow lumen surrounded by a single layer of polarized epithelial cells. However, ErbB2 activation prevents luminal clearance and induces luminal filling in acini formed in three-dimensional culture, and leads to activation of Akt, a known survival signal. Taken together these data indicate a potential role for ErbB2 at the apex of cell survival signaling via Src, FAK, and Akt, contributing to luminal cell survival in three-dimensional culture. We have thus confirmed Src, FAK, and Akt as potential players in early onset of breast cancer, and targeting these signaling players concurrently with ErbB2 may prove effective, especially in early stage breast cancers. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-08-31 16:25:59.917

Metastasis is the leading cause of mortality in human breast cancer. However, there are few predictive, prognostic, or therapeutic targets of breast cancer metastasis. Ezrin, a membrane cytoskeletal cross-linker, is frequently over-expressed in human breast cancer and is required for motility and invasion by cultured epithelial cells. Our group has recently shown that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in the transformation of epithelial cells, in which ezrin is phosphorylated on specific tyrosines, such as Y477, by Src (91, 93). We therefore examined whether Src/ezrin interaction also regulates invasion and metastasis of breast cancer. This thesis presents the following results: 1) In a murine system, ezrin and Src are differentially localized in nulliparous, lactating mammary glands and PyMT-induced tumours, with pronounced apical expression in nulliparous mammary glands but non-polarized strong cytoplasmic expression in PyMT-induced tumours. 2) Increased expression and activation of ezrin, Src and Met in PyMT-induced tumours compared to normal breast tissues was observed. A concomitant increased expression of activated Stat3 and HGF was also observed in PyMT-induced tumours, consistent with the establishment of an HGF/Met autocrine loop. 3) In invasive human breast tumours, from a premenopausal patient cohort, ezrin showed significantly greater cytoplasmic localization compared to non-neoplastic epithelial ducts in normal mammoplasties. 4) In a mouse breast carcinoma xenograft model, a Y477F ezrin mutant (not phosphorylatable by Src), significantly reduced local invasion of primary tumours and spreading into visceral organs, yet, it did not significantly affect primary tumour growth rate. 5) Y477F ezrin-expressing tumours exhibited focal areas of incomplete membranous ezrin staining which was absent in control tumours. Moderate/strong cytoplasmic ezrin staining was evident in both tumour groups. Thus, ezrin is differentially localized in non-invasive versus invasive mammary tumours. Our study implicates a role of the Src/ezrin pathway in regulating local invasion and metastasis of breast carcinoma cells and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic marker and treatment target for invasive breast cancer. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-04-28 12:24:25.286

No description available.

Apoptosis and cell survival are two seemingly opposing fate-determining processes that are regulated by distinct and complex signaling pathways. Caspase-8, an apical caspase, plays a pivotal regulatory role in initiating apoptosis. c-Src, a prototypical member of the Src family kinases (SFKs), regulates a myriad of cellular processes including cell mitogenesis, proliferation, growth and migration. Although the regulation of caspase-8 by c-Src has been suggested, the reciprocal regulation of these two seemingly opposing signaling molecules, caspase-8 and c-Src, has never been explored. To study this reciprocal regulation, we asked three questions. (1) Can active caspase-8 negatively regulate c-Src activity to allow the propagation of apoptosis? (2) Can c-Src negatively regulate caspase-8 activity to prevent the propagation of apoptosis? (3) Can caspase-8, when its enzymatic activity is inhibited, further promote c-Src activity to allow the propagation of cell survival? To address these questions, we first investigated the effect of active caspase-8 on the activation and activity of c-Src. We discovered that active caspase-8 inhibited c-Src activation and some of its downstream effectors. Next, we investigated whether c-Src could tyrosine phosphorylate caspase-8. We discovered that c-Src could phosphorylate caspase-8 at multiple tyrosine sites. We then examined whether tyrosine phosphorylated caspase-8 prevents apoptosis. We found that phosphorylation of caspase-8 at Y465 prevented its cleavage, and activity towards activating caspase-3 and towards causing cell morphological changes associated with apoptosis. Finally, we studied whether tyrosine phosphorylation of caspase-8 could further promote the activation of c-Src. We showed that phosphorylation of caspase-8 at both Y465 and Y397 resulted in the activation of c-Src and extracellular signal-regulated kinase 1/2 (Erk1/2). In conclusion, this work demonstrated the reciprocal regulation of two opposing signaling molecules, caspase-8 and c-Src. These results also suggest an elegant mechanism for a cell to commit efficiently and rapidly to a fate-determining process, either apoptosis or survival, by further suppression of the opposing signaling pathway.

The pathogenesis of diabetic nephropathy (DN) remains incompletely understood. In previous studies, we observed the activation of Tyr kinase Src by high glucose (HG) and showed that Src is required for MAPK activation and synthesis of collagen IV in cultured rat mesangial cells (MCs). Reactive oxygen species (ROS) are also important mediators of DN, and our present study aimed to investigate the role of Src in HG-induced ROS generation. In MCs, we found that HG led to ROS accumulation that was blocked by Src inhibitors or Src-specific siRNA. Downstream of Src, Vav2 was phosphorylated/activated leading to Rac1-dependent NADPH oxidase activation. Long-term HG exposure resulted in Src-dependent Nox4 protein induction. Nox2-specific siRNA abrogated ROS production only in short-term HG, while Nox4-specific siRNA blocked ROS production only in long-term HG. Taken together, our data indicate Src to be important in mediating ROS generation from both Nox2- and Nox4-containing NADPH oxidases.