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

In this study, luteinized human granulosa cells (GC) obtained during in vitro fertilization procedures were used as a model system to evaluate the effects of ethanol (EtOH), a well‐known reproductive toxin, on epidermal growth factor (EGF) and gonadotropin‐stimulated steroidogenesis. Our results demonstrate that the basal progesterone (P4) and estradiol (E2) secretion by human GC in vitro was dependent on the ovarian stimulation protocol. EGF significantly enhanced P4, but not E2, secretion in human GC from clomiphene citrate (CC), human menopausal gonadotropin (hMG), and hMG/gonadotropin‐releasing hormone agonist (GnRH‐a)‐treated patients. The effects of EGF plus luteinizing hormone (LH) were additive in cells from the CC group, but less than additive in hMG and hMG/GnRH‐a groups. EtOH at 20 mM or more inhibited EGF stimulated P4 secretion in human GC from all three patient groups. EtOH inhibited P4 secretion stimulated by EGF and LH cotreatment in the CC and hMG/GnRH‐a groups, but not in human GC from the hMG‐treated patients. These results suggest that basal and EGF or LH‐stimulated P4 secretion by human GC, as well as the effects of EtOH, are profoundly influenced by the follicle's hormonal milieu.

A new cycad species, Cycas fushunensis sp. nov., is described from the Lutetian Jijuntun Formation at Fushun Coalmine, Liaoning Province, northeast China, based on a well-preserved partial frond containing about 15 leaflets. The fossil is characterized by a single strong vein per leaflet, decurrent leaflet base and haplocheilic stomata, suggesting that the fossil is attributed to the genus Cycas of Cycadaceae. Epidermal anatomical comparisons between the fossil and 17 selected modern Cycas species further indicate that C. fushunensis sp. nov. closely resembles Cycas panzhihuaensis Zhou et Yang, an endemic cycad to southwest China, due to characters shared, such as the straight anticlinal walls of both adaxial and abaxial epidermal cells and granular to striate cuticular characters on the internal surface of guard cell periclinal walls. The occurrence of close-to-modern Cycas from the early Cenozoic largely casts doubt on a hypothesis of the late Miocene differentiation of modern cycads, suggested by a recent molecular phylogenetic study.

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.

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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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