Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed “reverse biopanning,” which can then be selected for further development based on their peptide receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in the cell surface in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be −360.645 kcal/mol, −487.239 kcal/mol, and −595.328 kcal/mol for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide in vitro via electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor.
Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes, were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules.Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery..
Targeted therapy remains one of the biggest challenges in the effective treatment of cancer. Due to their heterogeneity, not all tumors respond to a particular treatment regimen with similar efficacy. Thus, there is an urgent need to develop targeted therapies that are not only highly specific to cancer subtypes but also have minimal toxicity towards normal tissue. Currently, development of anti-cancer antibodies is limited to antigens that are either overexpressed or antigens that are only expressed in a few patients. This limits both the number of available targets and their selectivity for tumors. Using phage display technology, we have identified proteins that are induced by clinical doses of ionizing radiation (IR): tax-interacting protein 1 (TIP-1) and TATA box-associated factor 15 (TAF-15). Following IR treatment, cell surface expression of both TIP-1 and TAF-15 is enhanced in a number of human cancers, including brain, lung, breast and pancreatic cancer. The underlying premise is that IR induces a stress response resulting from DNA strand breaks and activation of ATM repair pathways. After IR stress, these antigens translocate to the surface where they can be targeted for therapeutic purposes. These radiation-inducible neoantigens present a new synergistic model for the treatment of cancer using IR. In this study, we characterized both radiation-inducible targets in vitro and in vivo. Western blotting, immunohistochemistry and flow cytometry data all demonstrated increased cell surface expression of both TIP-1 and TAF-15 after irradiation. We also developed a monoclonal antibody and a single chain antibody against TIP-1 and are currently in the process of developing antibodies against TAF-15. ELISA and near infra-red whole mouse imaging results demonstrate high affinity and high specificity of our lead anti-TIP-1 antibody and anti-TAF15 peptide in lung, brain, breast and pancreatic cancer. Dosimetry studies using immunofluorescence and Western blot analysis show a dose-dependent increase in expression of TIP-1 and TAF15, suggesting that these proteins may play a key role in the cancer cell's response to radiation stress. We are currently in the process of characterizing the mechanisms of how these proteins contribute to radioresistance and cancer survival processes such as proliferation, apoptosis and metastasis. Citation Format: Lincoln Muhoro, Heping Yan, Jeremy Hunn, Dinesh Thotala, Daniel Ferraro, Dennis Hallahan. Characterization and targeting of radiation-inducible neoantigens in multiple cancer types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4597. doi:10.1158/1538-7445.AM2014-4597
Poly-ethylene glycol (PEG), a hydrophobic polymer used to improve the circulation and biocompatibility of biomolecules, accumulates in solid tumors in a size-dependent manner that has been attributed to the enhanced permeability and retention (EPR) effect. Irradiation of cancer disrupts cancer microvasculature, resulting in enhanced EPR effect. In the present study, we found that radiation increases the retention of PEG in solid tumors. The accumulation of PEG in solid tumors was observed with in vivo imaging. Nude mice were prepared with heterotopic unilateral murine and human glioma (GL261, U87) and lung carcinoma (LLC, A549) tumors. A near-infrared (NIR) dye conjugated to PEG was then administered by IV to the mice which were imaged by NIR for seven days. There was increased retention of PEG-dye in tumors that had been irradiated. To further elucidate the mechanism of this increased retention, a similar in vivo imaging experiment was conducted in bilateral heterotopic tumor models (GL261, U87, LLC, A549) for seven days. The irradiated tumors demonstrated increased accumulation of PEG-dye, suggesting that the response to irradiation influenced the accumulation of PEG-dye. To observe the distribution of PEG-dye in the solid tumors, after the in vivo imaging was complete, the tumors were harvested and then sectioned for microscopy and histological staining. PEG-dye was observed in both the necrotic and non-necrotic regions of the tumor. The cancer microvasculature environment differs depending on where the tumor is located; to determine if the increased uptake was limited to only the hind limb, in vivo imaging was conducted in nude mice bearing orthotopic brain (GL261) and lung (LLC) tumors. PEG-dye was also observed to accumulate in these irradiated tumor models. Radiation induced EPR led to cancer specific delivery of PEG conjugates. Collectively, our data suggest that irradiation increases the retention of PEG-dye in solid tumors. This effect appears in more than one tumor type suggesting that the mechanism may arise from an increase in EPR following irradiation of cancer. If so, the concurrent administration of radiation therapy with polymer-conjugated therapeutic complexes may improve targeted drug delivery to cancer. Citation Format: Jerry Fong, Daniel J. Ferraro, Jeremy Hunn, Stefan Roberts, Mikhail Y. Berezin, Buck E. Rogers, Dennis E. Hallahan. Radiation increases permeability and retention of PEG-conjugates in solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2666. doi:10.1158/1538-7445.AM2013-2666
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