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Dans le traitement des cancers des voies aérodigestives supérieures (VADS), une approche biologique par des anti-EGFR (Epidermal Growth Factor Receptor) comme le cetuximab (Erbitux®) a récemment été proposée. Le cetuximab est un anticorps monoclonal chimérique qui se lie spécifiquement au domaine extracellulaire de l’EGFR, régulateur central de la prolifération et de la différenciation dans les cancers. Par cette liaison, le cetuximab entre en compétition avec les ligands du récepteur et empêche son activation, induit son internalisation et bloque la transduction du signal vers les voies de signalisation en aval. Même si cette approche thérapeutique est rationnelle puisque l’EGFR est surexprimé dans la plupart des cancers et notamment dans les cancers des VADS, certains types de cancers présentent une résistance à cet anticorps. Parmi les molécules qui ciblent EGFR il existe également des inhibiteurs de l’activité tyrosine kinase intracellulaire de l’EGFR comme le gefitinib (Iressa®), mais ce dernier n’est actuellement pas prescrit dans le traitement des cancers des VADS.Le but de notre travail a été d’étudier les mécanismes de résistance au cetuximab dans des lignées cellulaires de cancers des VADS puis de proposer des associations thérapeutiques pouvant pallier à cette résistance.Nous avons choisi deux lignées cellulaires de cancers des VADS, CAL33 et SQ20B en comparaison à la lignée épidermoïde A431 sur exprimant EGFR et sensible au cetuximab. Nous avons pu mettre en évidence que CAL33 et SQ20B étaient résistantes au cetuximab mais de manière surprenante sensibles au gefitinib. Nous avons montré que l’absence d’inhibition de phosphorylation d’AKT et qu’une altération de l’internalisation de l’EGFR par le cetuximab étaient responsables en partie de la résistance au cetuximab dans ces modèles cellulaires.Afin de pallier à cette résistance nous avons alors étudié les conséquences biologiques de l’association du cetuximab avec (i) des inhibiteurs de la voie PI3K/AKT par différentes approches et avec (ii) les radiations ionisantes. Dans un premier temps, nous avons étudié l’influence de l’inhibition de la voie AKT par un inhibiteur de PI3K ou un siRNA ciblant AKT. Nous avons démontré que l’inhibition de la phosphorylation d’AKT par l’inhibiteur LY294002 sensibilisait au cetuximab la lignée CAL33 porteuse d’une mutation activatrice du gène PIK3CA codant pour la sous-unité catalytique p110 de la protéine PI3K. Nous avons montré que la persistance de l’activation d’AKT dans la lignée CAL33 prévenait l’effet anti tumoral du cetuximab, tandis que la résistance au cetuximab dans la lignée SQ20B ne semblait pas dépendante de la voie AKT.Une association de traitement du cetuximab avec les radiations ionisantes est déjà proposée en clinique dans le traitement des cancers des VADS. Nous avons donc dans un second temps déterminé les effets de cette association de traitement dans les lignées SQ20B et CAL33 respectivement sauvage et mutée dans la voie de signalisation AKT et dans la lignée contrôle A431. Nous avons montré que l’association du cetuximab aux radiations ionisantes potentialisait l’effet du cetuximab sur l’inhibition de prolifération de la lignée A431 alors que nous n’avons observé aucune potentialisation de l’effet du cetuximab sur la prolifération dans les lignées résistantes CAL33 et SQ20B. Dans ce travail, nous montrons que la voie AKT apparaît donc comme un élément central dans la réponse au cetuximab dans la lignée CAL33 et que l’association du cetuximab avec un inhibiteur de la voie PI3K/AKT pourrait être une bonne option thérapeutique dans le traitement des cancers des VADS mutés pour PIK3CA. / In the treatment of HNSCC (Head and Neck Squamous Cell Carcinoma), a biological approach by anti-EGFR (Epidermal Growth Factor Receptor) such as cetuximab (Erbitux ®) has been recently proposed. Cetuximab is a chimeric monoclonal antibody that specifically binds the extracellular domain of EGFR, a central regulator of proliferation and differentiation in cancer. For this binding, cetuximab is in competition with EGFR ligands, prevents receptor activation, induces its internalization and blocks the transduction and the downstream signaling pathways. Although this therapeutic approach is rational because EGFR is overexpressed in most of cancers, some cancers are resistant to this antibody. Among the molecules that target EGFR, there are also inhibitors of intracellular tyrosine kinase activity of EGFR such as gefitinib (Iressa ®), but these molecules are not currently used for the treatment of HNSCC cancers. The aim of our study was to characterize resistance mechanisms to cetuximab in a panel of HNSCC cell lines and to propose the treatment combination able to overcome this resistance.We chose two cell lines of HNSCC, CAL33 and SQ20B in comparison with A431 cell line which over express EGFR and which is sensitive to cetuximab. We had shown that CAL33 and SQ20B were resistant to cetuximab but sensitive to gefitinib. This study showed that the absence of inhibition of phosphorylation of AKT by cetuximab and the alteration of the EGFR internalization were responsible in part of the cetuximab resistance in these cell models. Then we studied the biological effects of the combination of cetuximab with (i) inhibition of PI3K/AKT by different approaches and (ii) ionizing radiation.We initially studied the influence of inhibition of AKT pathway by a PI3K inhibitor or siRNA targeting Akt. We demonstrated that inhibition of AKT phosphorylation by LY294002 sensitized CAL33 to cetuximab. This cell line carries an activating mutation of the PIK3CA gene encoding the catalytic subunit p110 of PI3K protein. We have shown that persistent activation of AKT in line CAL33 warned antitumor effect of cetuximab, while resistance to cetuximab in line SQ20B did not seem dependent on the AKT pathway. A combination therapy of cetuximab with ionizing radiation is already proposed in the clinical treatment of these cancers. We therefore determined n the second step, the effects of this combination treatment in lines CAL33 and SQ20B respectively in the wild and mutant AKT signaling pathway and the control line A431. We have shown that the combination of cetuximab to ionizing radiation potentiated the effect of cetuximab on the inhibition of proliferation of line A431, whereas we observed no potentiation of the effect of cetuximab on the proliferation in the resistant lines CAL33 and SQ20B. In this work, we show that the AKT pathway is therefore a central element in the response to cetuximab in line CAL33 and the combination of cetuximab with an inhibitor of the PI3K/AKT pathway might be a good therapeutic option in the treatment of these cancers with a PIK3CA mutation.

We have previously demonstrated that Llgl1 loss results in a gain of mesenchymal phenotypes and a loss of apicobasal and planar polarity. We now demonstrate that these changes represent a fundamental shift in cellular phenotype. Llgl1 regulates the expression of multiple cell identity markers, including CD44, CD49f, and CD24, and the nuclear translocation of TAZ and Slug. Cells lacking Llgl1 form mammospheres, where survival and transplantability is dependent upon the Epidermal Growth Factor Receptor (EGFR). Additionally, Llgl1 loss allows cells to grow in soft-agar and maintain prolonged survival as orthotopic transplants in NOD-SCID mice. Lineage tracing and wound healing experiments demonstrate that mammosphere survival is due to enhanced EGF-dependent migration. The loss of Llgl1 drives EGFR mislocalization and an EGFR mislocalization point mutation (P667A) drives these same phenotypes, including activation of AKT and TAZ nuclear translocation. Together, these data indicate that the loss of Llgl1 results in EGFR mislocalization, promoting pre-neoplastic changes.

The discovery of the existence of the cell membrane has led to a search for its organization on a molecular scale. The advent of artificial lipid bilayers and the development of electron microscopy in the 1930's provided direct visual evidence for the existence of the cell membrane and drove forward models of membrane structure based its known composition of proteins, lipids and carbohydrates. The fluid mosaic model of membrane structure, based on thermo- dynamics and newly developed protein structural studies of the time, placed integral globular membrane proteins within a fluid phospholipid bilayer. This model allowed for the association of proteins into groups and the possible mobility of proteins within the lipid bilayer. At the the same time fluorescence microscopy demonstrated movement of proteins in the plane of the lipid bilayer. Since then experimental techniques have been developed that show protein complexes of varying sizes do exist and so this gives us the opportunity to ask how receptor proteins fit into the molecular organization of the cell membrane. This thesis presents an investigation into how the epidermal growth factor receptor (EGFR) organizes in the cell membrane of colorectal carcinoma cells. First a new cell line for studying the receptor by stably expressing the epidermal growth factor receptor conjugated to enhanced green fluorescent protein (EGFR-eGFP) in SW620 cells was developed. This is an interest- ing cell line because it originates from a colonic adenocarcinoma that during the process of metastasis has lost the ability to express the EGFR. It therefore provided an environment for the expression of the fluorescent form of the receptor more in keeping with its natural environment. The technique of total internal reflection fluorescence (TIRF) microscopy was used to visualize the fluorescently tagged receptor in the cell membrane. This technique uses the principles of total internal reflection to excite fluorescence in molecules located only 100 nm into the cell. Because sources of fluorescence from outside the illuminated area are minimized individual fluorescent molecules can be imaged. The spots in the images, produced by the fluorophores, were detected using a single molecule detection and tracking algorithm. The intensities of these detected spots were analysed and compared with that from a single molecule of enhanced green fluorescent protein (eGFP). This gave an estimate of the number of receptors contained within each receptor complex. Before ligand binding most of the receptors were found to be located in complexes containing up to eight molecules and most frequently they were found in complexes of two molecules. Larger complexes of receptors were found to have formed after activation of the receptor by its ligand.

The Epidermal Growth Factor Receptor (EGFR) is associated with a variety of cancers, including brain, lung, cervix, renal and breast. It is part of a family of receptors known as the ErbB receptors (ErbB1/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), transmembrane proteins found on epithelial cells responsible for a multitude of signaling events. In cancers, EGFR is frequently mutated or improperly expressed, upregulated in more than 50 percent of basal-like cancers. Mutations commonly promote constitutive activation or increase receptor recycling. In basal-like breast cancers such as triple negative breast cancer (TNBC), named for the lack of hormone receptors (estrogen and progesterone) and the HER2 receptor, EGFR is highly upregulated and associated with a variety of oncogenic activity, including increased proliferation and migration, and inhibition of cell death. Changes in these pathways are predicated on altered trafficking and activation of EGFR, events driven by variation in stimuli and interacting partners, such as other ErbB family members or oncogenic adaptor proteins such as MUC1, a member of the mucin family. In TNBC, upon stimulus with epidermal growth factor (EGF), EGFR colocalizes with MUC1 in intracellular vesicles distributed throughout the cytoplasm. These intracellular vesicles are associated with early endosomes, as indicated by the presence of early endosome antigen 1 (EEA1). Association with MUC1 prolongs the presence of EGFR in these vesicles, as EGFR's stay is significantly reduced in cells lacking MUC1. Retention in these vesicles by MUC1 inhibits trafficking of EGFR to the lysosome for degradation and is also associated with an increase in EGF-dependent migratory ability. Introduction of late endosome inhibitors (thereby preventing lysosomal targeting) increases migration in the absence of MUC1, the same effect as in the presence of MUC1. Further, inhibition of retrograde trafficking significantly decreases the rate of migration and changes cellular distribution of filopodia corresponding to migratory ability in MUC1-containing cells. Taken together, these data indicate that MUC1 is responsible for altering EGFR trafficking by retaining EGFR in EEA1-positive vesicles for prolonged periods, allowing for increased signal transduction through retrograde trafficking of EGFR and structural reorganization promoting a migratory phenotype. Loss of the polarity protein Llgl1 is associated with alterations in EGFR trafficking, promoting highly diffuse EGFR distribution throughout the cytoplasm versus along basolateral membranes. These changes in trafficking are also associated with increases in AKT and dual-phosphorylated-ERK signal transduction, both downstream targets of activated EGFR. Altering localization of EGFR to other membranes and intracellular vesicles without inducing polarity loss through a point mutation at amino acid 667 was found to also upregulate the AKT pathway. Mislocalization driven by polarity loss or point mutation in the basolateral targeting domain is sufficient to increase migration speeds of non-cancerous epithelial cell lines in vitro. This increased oncogenic activity is likely attributed to increased nuclear localization of the transcription factor TAZ (transcription co-activator with a PDZ-binding domain), whose nuclear translocation is associated with increased stem-like properties such as migration and survival. Together, these data reveal the oncogenic potential caused by alterations in EGFR trafficking that occur when polarity is lost or EGFR is improperly associated with proteins that promote changes to canonical EGFR localization and degradation, such as MUC1.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are highly effective therapies for sub-populations of non-small cell lung cancers. Specific mutations have been identified in the EGFR gene such as L858R which overstimulate cell pathways that lead to tumor growth. All tumors eventually develop resistance to this treatment, rendering them useless, and tumor growth progresses. Escape mutations in the EGFR gene were first seen in patients undergoing treatment with first-generation TKI erlotinib and gefitinib. T790M is a widely seen gate-keeping mutation which overcomes inhibition from erlotinib and gefitinib. Third-generation irreversible TKI, osimertinib, can inhibit tumor cells with this gate-keeping mutation thus overcoming a major hurdle in containment of tumor growth. Unfortunately, patients eventually develop resistance to osimertinib, exhausting options for managing non-small cell lung cancer. Here we analyzed H1975 cells which harbor L858R + T790M mutations. We aimed to track genomic, transcriptomic, and proteomic changes to uncover mechanisms cells use to develop resistance to osimertinib. We established cell colonies which were able to survive high dose treatment up to 2 µM osimertinib. We also saved cells with IC50 of 30 nM to represent drug-tolerant cells. We conducted single-cell sequencing of mRNA transcription and performed hierarchal gene analysis which identified CD74 as a novel factor which was upregulated in drug-tolerant cells. Further we showed CD74 gene was accessible as open chromatin for easy upregulation. Western blot analysis showed increased expression of CD74 after 24 hours of osimertinib treatment. Using siRNA in H1975 cells, we conducted knockdown experiments of CD74 during osimertinib treatment and showed reduced viability. Next, H1975 cells lines were engineered with deletions in CD74 to knockout its expression. These cells also showed reduced viability in the presence of osimertinib. Quantification of apoptosis using caspase-glo assays showed greater activation of apoptosis in cell populations without CD74 compared to normal H1975 cells. H1975-CD74 knockout cells also took longer to become resistant to osimertinib when compared with control. These results show the role of CD74 in helping tumor cells survive EGFR TKI treatment. / 2023-12-05T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes occurs as a result of the failure of pancreatic insulin-producing β cells. The preservation or renewal of β cells is a strategy that can prevent diabetes by targeted manipulation of mechanisms associated with autoimmune β cell destruction or β cell regeneration. ErbB signaling, specifically epidermal growth factor receptor (EGFR) signaling, is associated with cell survival, growth, and proliferation. Thus, we investigated the role of the ErbB inhibitor, mitogen-inducible gene 6 (mig6), in pancreas development and in the progression to diabetes. Using morpholino knockdown in a zebrafish model of development, we discovered that mig6 is required for the generation of α and β cells as well as the formation of the exocrine pancreas. We suspect that the loss of mig6 function causes premature differentiation of ductal progenitor cells, and acts as a switch between progenitor differentiation and endocrine transdifferentiation. Furthermore, we established a pancreas-specific mig6 knockout mouse that maintained glucose tolerance and had a higher β cell mass after chemically-induced β cell injury by way of increased β cell proliferation. Our data suggests that mig6 is required during pancreas development and may be employed as a switch to direct the production of new β cells, but that during adulthood, it is detrimental to the recovery of β cell mass, making it a therapeutic target for β cell preservation after the onset of diabetes.

Thesis advisor: Jianmin Gao / Protein-protein interactions (PPIs) are essential for all biological functions. Developing peptides that disrupt these PPIs is an avid research effort, as peptides possess several advantages over small molecules and monoclonal antibodies. Peptide phage display is a useful tool in identifying peptides for targeting PPIs. This technology displays up to 10^10 unique polypeptides on the surface of bacteriophage, which after several rounds of panning enriches high affinity peptide sequences towards a target protein. Phage display is classically done on immobilized discrete protein; however, we propose to use this technology to identify peptides ligands for overexpressed oncogenic proteins on live cells in-vitro. This is a more accurate representation of the therapeutic target landscape and resembles how the peptide will interact with the receptor in-vivo. Several groups have explored live cell panning, such as Ruoslahti et al. and Cieslewicz et al., and while they demonstrate the capabilities of in-vitro style phage display, there are areas for improvement. We intend to improve on this previous work by 1. Identifying a peptide ligand against specific receptor/protein, and 2. By incorporating the use of covalent phage libraries to elucidate a high affinity binder. This work will be accomplished using the mammalian epidermal oncogenic cell line, A431, that is known to overexpress epidermal growth factor receptor (EGFR). Epidermal growth factor receptor (EGFR) is responsible for cellular proliferation, survival, differentiation and metastasis, which makes it an attractive target to inhibit oncogenic proliferation. Despite successfully marketed monoclonal antibodies and tyrosine kinase inhibitors, EGFR can mutate and develop resistance as diseases progress; this phenomenon, in addition to the benefits of peptides as therapeutics, are driving factors for pursuing this project. Despite our best efforts using non-covalent phage libraries to identify a viable ligand, screening against EGFR extracellular domain (ECD) has proven to be more difficult than anticipated. We hypothesize that non-covalent phage libraries do not possess any sequences with a high enough binding affinity for this protein, and that the use of covalent libraries will be needed to pull out a positive hit. Due to these findings, we have successfully constructed two phage libraries, a ACX7C and a ACX7C-TEV, where the latter introduced a TEV protease cleavage site on the C’-terminal side of the randomized amino acids suitable for covalent warhead modification and screening. Further, we have begun work on constructing an EGF-displaying phage construct to aid in optimizing a live cell panning protocol. In the future, we plan to evaluate ligand affinity and protein density, as well as determine the optimal covalent warhead/peptide combination for live cell screenings. With this information, we intend to apply this to other oncogenic cell lines, such as MCF-10CA1a, to identify potent peptide ligands for overexpressed oncogenic proteins. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares