Gastric cancer ranks the fifth most common and third leading cause of cancer‐related deaths worldwide. Alterations in the RTK/MAPK, WNT, cell adhesion, TP53, TGFβ, NOTCH, and NFκB signaling pathways could be identified as main oncogenic drivers. A combination of altered pathways can be associated with molecular subtypes of gastric cancer. In order to generate model systems to study the impact of different pathway alterations in a defined genetic background, we generated three murine organoid models: a RAS‐activated (KrasG12D, Tp53R172H), a WNT‐activated (Apcfl/fl, Tp53R172H), and a diffuse (Cdh1fl/fl, Apcfl/fl) model. These organoid models were morphologically and phenotypically diverse, differed in proteome expression signatures and possessed individual drug sensitivities. A differential vulnerability to RTK/MAPK pathway interference based on the different mitogenic drivers and according to the level of dependence on the pathway could be uncovered. Furthermore, an association between RTK/MAPK pathway activity and susceptibility to HDAC inhibition was observed. This finding was further validated in patient‐derived organoids from gastric adenocarcinoma, thus identifying a novel treatment approach for RTK/MAPK pathway altered gastric cancer patients.

BACKGROUND:General, breed- and diet-dependent associations between feed efficiency in beef cattle and single nucleotide polymorphisms (SNPs) or haplotypes were identified on a population of 1321 steers using a 50K SNP panel. Genomic associations with traditional two-step indicators of feed efficiency - residual feed intake (RFI), residual average daily gain (RADG), and residual intake gain (RIG) - were compared to associations with two complementary one-step indicators of feed efficiency: efficiency of intake (EI) and efficiency of gain (EG). Associations uncovered in a training data set were evaluated on independent validation data set. A multi-SNP model was developed to predict feed efficiency. Functional analysis of genes harboring SNPs significantly associated with feed efficiency and network visualization aided in the interpretation of the results.RESULTS:For the five feed efficiency indicators, the numbers of general, breed-dependent, and diet-dependent associations with SNPs (P-value<0.0001) were 31, 40, and 25, and with haplotypes were six, ten, and nine, respectively. Of these, 20 SNP and six haplotype associations overlapped between RFI and EI, and five SNP and one haplotype associations overlapped between RADG and EG. This result confirms the complementary value of the one and two-step indicators. The multi-SNP models included 89 SNPs and offered a precise prediction of the five feed efficiency indicators. The associations of 17 SNPs and 7 haplotypes with feed efficiency were confirmed on the validation data set. Nine clusters of Gene Ontology and KEGG pathway categories (mean P-value<0.001) including, 9nucleotide binding / ion transport, phosphorous metabolic process, and the MAPK signaling pathway were overrepresented among the genes harboring the SNPs associated with feed efficiency.CONCLUSIONS:The general SNP associations suggest that a single panel of genomic variants can be used regardless of breed and diet. The breed- and diet-dependent associations between SNPs and feed efficiency suggest that further refinement of variant panels require the consideration of the breed and management practices. The unique genomic variants associated with the one- and two-step indicators suggest that both types of indicators offer complementary description of feed efficiency that can be exploited for genome-enabled selection purposes.

Introduction: Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands. Methods: The CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model. Results: JWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on G alpha(i) signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling. Conclusion: The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.

Metastatic melanoma is an aggressive, incurable cancer with historically few therapeutic options. The discovery that 60% of melanomas harbor the oncogenic BRAF_V600E mutation, which constitutively activates the MAPK pathway, has provided a promising new therapeutic axis. Although MAPK pathway inhibitor therapy has shown striking clinical results in BRAF_V600-mutant melanoma, this approach faces three limitations. First, 10-20% of BRAF_V600-mutant melanomas never achieve meaningful response to MAPK pathway inhibitor therapy (intrinsic resistance). Second, among BRAF_V600-mutant melanomas initially responding to MAPK pathway inhibitor therapy, relapse is universal (acquired resistance). Third, approximately 40% of melanomas lack BRAF_V600 mutations and so are not currently candidates for MAPK pathway inhibitor therapy. We sought to address each of these problems: by characterizing the phenomenon of intrinsic MAPK pathway inhibitor resistance, by finding ways to perturb mechanisms of acquired MAPK pathway inhibitor resistance, and by identifying novel dependencies in melanoma outside of the MAPK pathway. Intriguingly, the NF-kappa B pathway emerged as a common theme across these investigations. In particular, we establish that MAPK pathway inhibitor sensitive and resistant melanomas display distinct transcriptional signatures. Unlike most BRAF_V600-mutant melanomas, which highly express the melanocytic lineage transcription factor MITF, MAPK pathway inhibitor resistant lines display low MITF expression but high levels of NF-kappa B signaling. These divergent transcriptional states, which arise in melanocytes from aberrant MAPK pathway activation by BRAF_V600E, remain plastic and mutually antagonistic in established melanomas. Together, these results characterize a dichotomy between MITF and NF-kappa B cellular states as a determinant of intrinsic sensitivity versus resistance to MAPK pathway inhibitors in BRAF_V600-mutant melanoma. In separate investigations, we have shown that, NFKB1 p105, a member of the NF-kappa B family, intimately regulates levels of COT, a known effector of resistance to MAPK pathway inhibitors. Moreover, we have used shRNA screening to nominate particular nodes within the NF-kappa B pathway, including MYD88 and IRF3, as candidate melanoma lineage-specific dependencies. Cumulatively, although these studies use diverse approaches to investigate the limitations of MAPK pathway inhibitor therapy in melanoma, they converge in nominating the NF-kappa B pathway as a previously underappreciated feature of melanoma biology and suggest the relevance of this pathway for future investigation.

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses.

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.

Fibroblast growth factor (FGF) signalling plays many fundamentally important roles during the development of the mammalian embryo. However, its effects on pluripotent stem cells derived from mouse and human embryos appear to be markedly different. FGF2 is routinely added to culture medium for propagating undifferentiated human (hES) cells, whereas in mouse (mES) cell cultures FGFs have been described as regulators of their differentiated progeny. To assess the effect of FGF signalling on undifferentiated mES cells, the effects of FGF2 and 4 were analysed in the presence of saturating and sub-saturating levels of the inhibitor of differentiation, leukaemia inhibitory factor (LIF). Mouse ES cell self-renewal was quantified by measuring the expression of the stem cell specific reporter Oct4-LacZ in biochemical and fluorometric assays. Treatment with FGF reduced the expression of the OCT4-LacZ reporter, even under saturating concentrations of LIF and this was mirrored by decreased levels of OCT4 protein. Furthermore, treatment with FGF leads to upregulation of the ectodermal differentiation marker Pax6. These results suggest that FGF signalling has a direct impact on undifferentiated mES cells, and actively promotes their differentiation. To asses the effect of FGF signalling on hES cells without the influence of undefined factors, a feeder and serum free system was developed. Cells growing in this conditions for >20 passages maintained expression of surface (SSEA3 and TRA1-60 and 81) and internal (OCT4) markers specific for undifferentiated hES cells. Expression of these markers was dependant on the continuous presence of FGF2. Indeed, withdrawal of FGF2 resulted in a rapid decrease of in hES cell growth and of the emergence of cell flattened morphology and of the surface marker SSEA1, changes typically associated with differentiation. Two important signals activated by FGF in hES cells are the ERK/MAPK and PI3K pathways. To assess their functional relevance, hES cell cultures were treated with the drugs UO126 and LY294002, inhibitors of the MAPK and PI3K pathways respectively. Drug mediated suppression of the phosphorylation of these pathways, correlated with a reduction in cell growth, flattening of the colonies and reduction in SSEA4 expression. Use of SB431542, specific inhibitor of TGFβ/activin type I receptor kinase (Alk5) also resulted in the flattening of the colonies and the appearance of dispersed cells. Therefore, inhibition of MAPK and PI3K appears to impair growth and self-renewal in hES cells and this may be happening in conjunction with TGFβ/Activin pathway. Taken together, these results suggest that FGF signalling has opposite effects in mouse and human ES cells: inducing differentiation in mES and sustaining self-renewal in hES.

L'activation du récepteur AT[indice inférieur 2] de l'angiotensine II (Ang II) est associée à différentes réponses cellulaires dont l'inhibition de prolifération, le contrôle de l'apoptose et l'induction de la différenciation. Au cours du développement, le récepteur AT[indice inférieur 2] est fortement présent dans les tissus foetaux mais son expression chute drastiquement, quelques heures après la naissance. Chez l'adulte, seulement quelques tissus expriment ce récepteur (cellules glomérulées de la surrénale, utérus, cellules granulosa de l'ovaire et certaines zones du cerveau) mais sa ré-expression peut être observée au cours de certaines conditions pathologiques (défaillance cardiaque ou rénale, dommages tissulaires, lésions du système nerveux central). Ces observations suggèrent que le récepteur AT[indice inférieur 2] joue un rôle important au cours du développement, dans les processus de réponses aux blessures et dans les mécanismes d'adaptation. Dans les cellules NG108-15, l'activation du récepteur AT[indice inférieur 2] par l'Ang II induit la différenciation neuronale (Laflamme et al . 1996). Puisque les cibles intracellulaires du récepteur AT[indice inférieur 2] sont peu connues, le but de nos études était de déterminer les mécanismes d'action associés à son activation dans l'induction de l'élongation des neurites. Le récepteur AT[indice inférieur 2] n'est couplé à aucun des seconds messagers classiques (AMPc, production d'InsPs, Ca[indice supérieur 2+] ). Les effets connus du récepteur AT[indice inférieur 2] sont une augmentation ou une diminution des niveaux de monoxyde d'azote (NO) et de GMPc et, selon les modèles cellulaires et les conditions de culture utilisés, il peut activer ou inhiber les phosphatases et les p42/p44[indice supérieur mapk] en plus de modifier l'excitabilité membranaire (inhibition des courants calciques et activation des canaux potassiques). Dans les cellules NG108-15, nous avons trouvé que l'activation du récepteur AT[indice inférieur 2] par l'Ang II induit l'activation des p42/p44[indice supérieur mapk] par un mécanisme indépendant de la petite protéine G p21[indice supérieur ras] , un processus essentiel à l'induction de l'élongation des neurites (Gendron et al . 1999). Les travaux présentés dans le cadre de cette thèse montrent que la production de NO, suite à l'activation du récepteur AT[indice inférieur 2] par l'Ang II, est impliquée dans l'induction de la différenciation neuronale. Nous avons en effet observé que l'Ang II, par un mécanisme dépendant des protéines G[alpha indice inférieur i] , mène à une augmentation rapide des niveaux intracellulaires de GMPc, un second messager impliqué dans l'élongation et dans le branchement neuritique des cellules NG108-15. Bien que cette voie est essentielle à la différenciation neuronale, nous avons trouvé qu'elle n'est pas impliquée dans l'activation des p42/p44[indice supérieur mapk] .L'activation des p42/p44[indice supérieur mapk] par l'Ang II, qui est Ras- et NO-indépendante, est plutôt induite par une voie alternative impliquant les protéines Rap1 et B-Raf.L'application d'Ang II dans les cellules NG108-15 mène en effet à l'activation rapide de Rap1 (1-5 min) et de B-Raf (5-15 min), événement essentiel à la fois pour l'activation des p42/p44[indice supérieur mapk] et pour l'induction de la différenciation des cellules NG108-15. Finalement, nous avons montré que l'activation de cette voie se fait par un mécanisme indépendant de l'AMPc et de la PKA. Ensemble, nos résultats montrent que le récepteur AT[indice inférieur 2] active les voies nNOS/NO/GCs/GMPc et Rap1/B-Raf/MEK/MAPK, et que ces cascades participent de façon parallèle, à l'induction de la différenciation neuronale des cellules NG108-15, par un mécanisme indépendant de l'AMPc et de la PKA.

Une augmentation de la génération des dérivés réactifs de l’oxygène (DRO), tels que le peroxyde d’hydrogène (H2O2), joue un rôle clé dans la pathophysiologie des maladies cardiovasculaires (MCV). La croissance et la prolifération excessives des cellules musculaires lisses vasculaires (CMLV) ont été suggérées comme étant les mécanismes à la base de la dysfonction vasculaire. Une implication potentielle du facteur de transcription Early growth response protein-1 (Egr-1) dans le développement des dommages vasculaires a été proposée. Des études ont démontré que le H2O2 augmente l’expression de l’Egr-1 dans les CMLV. Cependant, les voies de signalisation intracellulaire menant à l’expression de l’Egr-1 en réponse au H2O2 restent à établir. L’objectif de cette étude vise à examiner les différentes voies de signalisation impliquées dans l’expression de l’Egr-1 induite par le H2O2 dans les CMLV. Le H2O2 augmente l’expression de l’Egr-1 en fonction du temps et de la dose dans les CMLV A10. Le blocage pharmacologique des tyrosines kinases insulin-like growth factor-1 receptor (IGF-1R) et c-Src, par AG1024 et PP2 respectivement, atténue l’expression de l’Egr-1 induite par le H2O2, alors que l’AG1478, un inhibiteur de l’epidermal growth factor receptor (EGFR), et le PP3, l’analogue inactif du PP2, n’ont aucun effet sur l’expression de l’Egr-1. Le blocage pharmacologique de l’extracellular signal-regulated kinase 1/2 (ERK1/2), par UO126, et de la protéine kinase C (PKC), par rottlerin et rö-31-8220, diminue l’expression de l’Egr-1 induite par le H2O2. En résumé, nos résultats suggèrent que le H2O2 déclenche l’expression de l’Egr-1 via l’IGF-1R, la kinase c-Src, l’ERK1/2 et la PKC dans les CMLV. / Increased generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), plays a key role in the pathophysiology of cardiovascular diseases (CVD). Excessive growth and proliferation of vascular smooth muscle cells (VSMCs) has been suggested as an important contributor of vascular dysfunction. A potential involvement of early growth response protein-1 (Egr-1), a zinc-finger transcription factor, in the development of vascular injury has been proposed. Recent studies have shown that H2O2 increases Egr-1 expression in VSMCs. However, signaling events leading to H2O2-induced Egr-1 expression are not fully understood. Therefore, this study aims to examine the signaling pathways implicated in H2O2-induced Egr-1 expression in VSMC. H2O2 increased Egr-1 expression in a time and dose-dependent fashion in A10 VSMC. Pharmacological blockade of tyrosine kinases insulin-like growth factor-1 receptor (IGF-1R) and c-Src, by AG1024 and PP2 respectively, attenuated H2O2-induced Egr-1 expression, while AG1478, an epidermal growth factor receptor (EGFR) inhibitor, and PP3, the inactive analogue of PP2, have no effect on Egr-1 expression. Pharmacological blockade of extracellular signal-regulated kinase 1/2 (ERK1/2), by UO126, and proteine kinase C (PKC), by rottlerin and rö-31-8220, decreased H2O2-induced Egr-1 expression. In summary, our results suggest that H2O2 triggers Egr-1 expression through IGF-1R, c-Src, ERK1/2 and PKC in VSMC.

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