Browsing by Subject "EGFR"

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Avidity effects of MinE07, an anti-EGFR aptamer, on binding to A431 cells
(2010) Avutu, Viswatej; Andrew D. Ellington
Cell growth, differentiation, and proliferation are all carefully regulated processes. Disruptions in these processes are often associated with malignant tumors. The epidermal growth factor receptor (EFGR), part of the ErbB family of receptors, is known to play a pivotal role in regulating numerous cell growth processes including morphology, differentiation, proliferation, and apoptosis in certain cell types. Overexpression or elevated levels of EGFR activity is associated with many different types of cancers. Numerous targeted anti-EGFR therapies have been developed, including monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors. Apatmers provide an attractive alternative to monoclonal antibodies due to their ease of synthesis and lack of immunogenicity. J18 and E07 are two aptamers which were selected for against EGFR. Due to 2’-fluoro pyrimidines modification, E07 was chosen for in vivo applications. E07 was further remodeled to a minimal length construct that still retained binding affinity for EGFR. A cell growth assay using E07 and the anti-EGFR mAb, Cetuximab, revealed that a much larger dose of aptamer was needed to achieve the same level of growth inhibition as Cetuximab. In an effort to improve the efficiency of E07, an experiment was designed to improve the Kd of E07 with avidity effects – supradditive effects observed upon dimerization or multimerization of monomers. Having been observed with peptides, it was hypothesized that nucleic acids might also display such avidity effects. Five dimeric constructs of the minimized E07 (MinE07) aptamer were tested using flow cytometry assays on A431 cells. Two variables were also tested: the orientation of the monomers in the dimeric construct and the distance separating the two monomers. Constructs were assembled in three different schemes. First, DNA organizers containing fluorophores (fluorescein) were used to direct formation. In a second strategy, the extensions added to MinE07 directed the formation of the dimer. Lastly, the dimer was created via transcription off of an ssDNA template. FACS data revealed that none of the constructs significantly produced avidity effects. However, Construct 3 did inconsistently demonstrate slight avidity effects. Depending on the conditions of the A431 cells, the cell surface and subsequent assays can change dramatically. The head-totail orientation proved to be more promising in permitting avidity effects. Because no significant avidity effects were seen, the effect of intra-aptamer distance on binding affinity could not truly be studied.
Dietary energy balance modulates growth factor signaling during multistage epithelial carcinogenesis in mouse skin
(2010-12) Moore, Tricia Wallace; DiGiovanni, John; Hursting, Stephen; Kline, Kimberly; deGraffenried, Linda; Fischer, Susan; Vasquez, Karen
Energy balance refers to the relationship between energy intake and energy expenditure. Epidemiological studies have established a clear association between energy balance and cancer, however the underlying mechanisms are unclear. The objective of the current study was to evaluate the impact of caloric consumption on epithelial carcinogenesis and identify potential mechanisms of inhibition or enhancement. Using ICR female mice, we demonstrated that positive energy balance enhanced, while negative energy balance inhibited susceptibility to multistage carcinogenesis in mouse skin. We next evaluated diet-induced changes in the epidermal proliferative response. Calorie restriction (CR) significantly reduced epidermal hyperproliferation, in the presence and absence of tumor promotion, as compared to diet-induced obesity (DIO). Additional studies were conducted to determine the impact of dietary manipulation on TPA-induced growth factor signaling. CR reduced, while DIO increased insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation, which subsequently modulated signaling downstream to Akt and mTOR. These diet-induced changes in growth factor signaling were confirmed under steady-state conditions in multiple epithelial tissues (i.e., skin, liver and dorsolateral prostate) in multiple mouse strains (FVB/N, C57BL/6 and ICR). Further analyses demonstrated that caloric consumption directly correlated with levels of cell cycle progression related proteins and inversely correlated with levels of cell cycle inhibitory proteins. Genetic reduction of circulating IGF-1, liver IGF-1 deficient (LID) mouse model, inhibited two-stage skin carcinogenesis, reduced epidermal hyperproliferation and attenuated IGF-1R and EGFR growth factor signaling during tumor promotion, similar to CR, suggesting a potential for IGF-1R and EGFR crosstalk. Further studies, demonstrated that IGF-1 induced EGFR activation in cultured mouse keratinocytes, possibly due to IGF-1R and EGFR heterodimerization or IGF-1 induced changes in EGFR mRNA expression. In vivo, CR reduced, while DIO increased IGF-1R and EGFR association during tumor promotion. Furthermore, CR attenuated EGFR ligand mRNA expression both in the presence and absence of TPA treatment. Collectively, these findings suggest that dietary energy balance modulates epithelial carcinogenesis, at least in part due to diet-induced changes in levels of circulating IGF-1, which then modulate IGF-1R and EGFR crosstalk and downstream signaling to cell cycle related proteins, subsequently altering epidermal hyperproliferation.
Interactions of composite gold nanoparticles with cells and tissue : implications in clinical translation for cancer imaging and therapy
(2012-12) Tam, Justina Oichi; Sokolov, Konstantin V. (Associate professor)
Current methods to diagnose and treat cancer often involve expensive, time-consuming equipment and materials that may lead to unwanted side effects and may not even increase a patient’s chance of survival. Thus, for a while now, a large part of the research community has focused on developing improved methods to detect, diagnose, and treat cancer on the molecular scale. One of the most recently discovered methods of cancer therapy is targeted therapy. These targeted therapies have potential to provide a patient with a form of personalized medicine because these therapies are biological molecules that specifically target other molecules involved with a cancer’s growth. Past trials using these therapeutic molecules, however, have led to controversial results, where certain patients responded better than others to the therapy for unknown reasons. Elucidating the reason behind these mixed results can be accomplished using metal nanoparticle technologies which could provide a bright signal to monitor the path that these therapeutic molecules take in vivo as well as enhance the molecule’s efficacy. Literature has shown that presenting targeting molecules in a dense manner to their target will increase these molecules’ binding affinity. This concept has been explored here to increase binding affinity of therapeutic molecules by attaching these molecules in a dense manner on the surface of gold nanoparticles, and correlating this increased affinity with therapeutic efficacy. Additionally, gold nanoparticles provide an easy surface for molecules to be functionalized on and have shown to be effective imaging, x-ray, and photothermal therapy agents. A major roadblock to using these gold nanoparticles clinically is their non-degradability and thus potential to cause long-term negative side effects in vivo. A platform for developing biodegradable gold nanoparticles is also explored here to take advantage of the gold nanoparticles’ excellent imaging and drug delivery capabilities while still allowing them to be used safely in the long term.
The effects of dimer and oligomer separation on cell signaling in the EGFR family
(2020-02-05) Pattengale, Sarah Ruth; Leahy, Daniel J. (Ph. D. in biophysics)
The EGFR family of receptor tyrosine kinases (RTKs) includes EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB. The EGFR family members play crucial roles in development and in regulating normal cellular processes. However, these receptors are found to be overexpressed or mutated in various cancers. The complete mechanism by which the EGFR family is autoregulated is still unknown. This work focuses on the role of the extracellular domain of EGFR family members on receptor activation, dimerization/oligomerization, and downstream signaling. We generated a system using designed ankyrin repeat proteins (DARPins) to study EGFR extracellular domain dimers of varying separation distances. We biochemically, cellularly, and structurally characterize interactions between HER3 and A30, an RNA that binds to the extracellular domain of HER3 and reduces HER2 phosphorylation. This work also includes preliminary efforts to characterize HER2-HER3 oligomers using single molecule photobleaching experiments. This work lays the groundwork for future studies involving EGFR family receptor regulation