Breast cancer is the leading cause of cancer-related deaths of women in the united states. Traditionally, Breast cancer is predominantly treated by a combination of surgery, chemotherapy, and radiation therapy. However, due to the significant negative side effects associated with these traditional treatments, there has been substantial efforts to develop alternative therapies to treat cancer. One such alternative therapy is a peptide-based therapeutic cancer vaccine. Therapeutic cancer vaccines enhance an individual's immune response to a specific tumor. They are capable of doing this through artificial activation of tumor specific CTLs (Cytotoxic T Lymphocytes). However, in order to artificially activate tumor specific CTLs, a patient must be treated with immunogenic epitopes derived from their specific cancer type. We have identified that the tumor associated antigen, TPD52, is an ideal target for a therapeutic cancer vaccine. This designation was due to the overexpression of TPD52 in a variety of different cancer types. In order to start the development of a therapeutic cancer vaccine for TPD52-related cancers, we have devised a two-step strategy. First, we plan to create a list of potential TPD52 epitopes by using epitope binding and processing prediction tools. Second, we plan to attempt to experimentally identify MHC class I TPD52 epitopes in vitro. We identified 942 potential 9 and 10 amino acid epitopes for the HLAs A1, A2, A3, A11, A24, B07, B27, B35, B44. These epitopes were predicted by using a combination of 3 binding prediction tools and 2 processing prediction tools. From these 942 potential epitopes, we selected the top 50 epitopes ranked by a combination of binding and processing scores. Due to the promiscuity of some predicted epitopes for multiple HLAs, we ordered 38 synthetic epitopes from the list of the top 50 epitope. We also performed a frequency analysis of the TPD52 protein sequence and identified 3 high volume regions of high epitope production. After the epitope predictions were completed, we proceeded to attempt to experimentally detected presented TPD52 epitopes. First, we successful transduced parental K562 cells with TPD52. After transduction, we started the optimization process for the immunoprecipitation protocol. The optimization of the immunoprecipitation protocol proved to be more difficult than originally believed and was the main reason that we were unable to progress past the transduction of the parental cells. However, we believe that we have identified the issues and will be able to complete the experiment in the coming months.

The p53 gene functions as a tumor suppressor that inhibits proliferation, regulates apoptosis, DNA repair, and normal cell cycle arrest. Mutation of the p53 gene is linked to be prevalent in 50% of all human cancers. In this paper, we are exploring triple negative breast cancer and the effects of simvastatin on tumor growth and survival. Simvastatin is a drug that is primarily used to treat high cholesterol and heart disease. Simvastatin is unique because it is able to inhibit protein prenylation through regulation of the mevalonate pathway. This makes it a potential targeted drug for therapy against p53 mutant cancer. The mechanism behind this is hypothesized to be correlated to aberrant activation of the Ras pathway. The Ras subfamily functions to transcriptionally regulate cell growth and survival, and will therefore allow for a tumor to thrive if the pathway is continually and abnormally activated. The Ras protein has to be prenylated in order for activation of this pathway to occur, making statin drug treatment a viable option as a cancer treatment. This is because it acts as a regulator of the mevalonate pathway which is upstream of protein prenylation. It is thus vital to understand these pathways at both the gene and protein level in different p53 mutants to further understand if simvastatin is indeed a drug with anti-cancer properties and can be used to target cancers with p53 mutation. The goal of this project is to study the biochemistry behind the mutation of p53's sensitivity to statin. With this information we can create a possible signature for those who could benefit from Simvastatin drug treatment as a possible targeted treatment for p53 mutant cancers.

Adrenocortical carcinoma (ACC) is a rare and deadly disease that affects 0.5-2 people per million per year in the US. Currently, the first line clinical management includes surgical resection, followed by treatment with the chemotherapeutic agent mitotane. These interventions, however, have limited effectiveness, as the overall five-year survival rate of patients with ACC is less than 35%. Therefore, further scientific investigation underlying the molecular mechanisms and biomarkers of this disease is of high importance. The aim of this project was to identify potential biomarkers that may be used as prognosticators as well as candidate genes that might be targeted to develop new therapies for patients with ACC. An analysis of publicly-available datasets revealed PDZ-binding kinase (PBK) as being upregulated roughly 9-fold in ACC tissue compared to normal adrenal tissue. PBK has been implicated as an oncogene in several other systems, and its expression has been shown to negatively impact patient survival. Initial experiments have confirmed the upregulation of PBK in H295R cells, a human ACC cell line. We effectively silenced PBK (>95% reduction in protein content) in H295R cells using lentiviral shRNA constructs. Using high and low PBK expressing cells, we performed soft agar assays for colony formation, and found that the PBK-silenced cells produced two-fold fewer colonies than the vector control (p<0.05). This indicates that PBK likely plays a role in tumorigenicity. We further conducted functional studies for apoptosis and proliferation to elucidate the mechanism by which PBK increases tumorigenicity. Preliminary results from MTS assays showed that after 9 days, PBK-silenced cells proliferated significantly less than the vector control, so PBK likely increases proliferation. Together these data identify PBK as a kinase implicated in ACC tumorigenesis. Further in vitro and in vivo studies will be conducted to evaluate PBK as a potential therapeutic target in adrenocortical carcinoma.

Schizophrenia risk is influenced by both genetic and environmental factors. The immediate early gene early growth response 3 (Egr3), is regulated downstream of several schizophrenia risk genes and encodes a zinc-finger transcription factor protein. Previous studies from our lab indicate that Egr3 deficient (Egr3 -/-) mice exhibit schizophrenia-like phenotypes. We also discovered decreased serotonin 2a receptors (5-HT2AR) in the Egr3 -/- mice, similar to studies that reported decreased 5-HT2ARs in schizophrenia patients. We previously reported that sleep deprivation, a mild stress, causes the over expression of Egr3 and the serotonin 2a gene (Htr2a) in the cortex. To determine whether EGR3, a transcription factor, regulates Htr2a in the prefrontal cortex after sleep deprivation, Egr3 -/-and Egr3 +/+ mice were sleep deprived for eight hours. Transgenic mice were used that expressed enhanced green fluorescent protein (EGFP) under control of the Htr2a promoter via a bacterial artificial chromosome (BAC). Immunohistochemistry was performed to identify EGFP containing cells. Data analysis revealed no significant interaction between genotype and sleep deprivation in 5-HT2AR/EGFP containing cells within the prefrontal cortex. Based on the findings of this study, more data is needed to better determine the relationship between sleep deprivation and its effect on the regulation of Htr2a through in an EGR3 dependent manner.

As advanced as current cancer therapeutics are, there are still challenges that need to be addressed. One of them is the non-specific killing of normal cells in addition to cancerous cells. Ideal cancer therapeutics should be targeted specifically toward tumor cells. Due to the robust self-assembly and versatile addressability of DNA-nanostructures, a DNA tetrahedron nanostructure was explored as a drug carrier. The nanostructure can be decorated with various molecules to either increase immunogenicity, toxicity, or affinity to a specific cell type. The efficiency of the specific binding and internalization of the chosen molecules was measured via flow cytometry. Using a murine B cell lymphoma as the model system, several targeting molecules have been evaluated for their specific binding and induced internalization of DNA nanostructures, including an anti-Igκ antibody, an idiotype-binding peptide, and a g-quadruplex nucleolin specific aptamer. It was found that adding the anti-Igκ antibody appeared to provide increased binding and facilitated cellular internalization. Also, it was found that the presence of CpG appeared to aid in the binding of nanostructures decorated with other molecules, as compared to nanostructures without CpG. The g-quadruplex aptamer thought to specifically bind cancer cells that overexpress nucleolin was tested and found to have better binding to cells when linked to the nanostructure than when alone. The drug doxorubicin was used to load the DNA-nanostructure and attempt to inhibit cancer cell growth. The DNA-nanostructure has the benefit of being self-assembled and customizable, and it has been shown to bind to and internalize into a cancer cell line. The next steps are to test the toxicity of the nanostructure as well as its specificity for cancerous cells compared to noncancerous cells. Furthermore, once those tests are completed the structure’s drug delivery capacity will be tested in tumor bearing mice. The DNA-nanostructure exhibits potential as a cancer specific therapeutic.

Measles and mumps are highly contagious, vaccine-preventable diseases with cases continuing to persist in high two-dose vaccinated populations. Recent outbreaks on university and college campuses across the United States prompt a need for further understanding of the immunity levels afforded by the MMR vaccine which has significantly decreased incidence rates of measles and mumps since it was introduced.
Current methods for IgG antibody detection include enzyme immunoassays (EIA) such as the commercially available Diamedix Immunosimplicity® Measles IgG test kit and the Diamedix Immunosimplicity® Mumps IgG test kit. EIAs generally provide high sensitivity and strong specificity, however, there is a need for rapid screening of measles and mumps specific immunity in outbreak and resource-limited areas which could be solved by use a point-of-care (POC) platform.
This study aims to optimize a point-of-care device for the multiplexed detection of MeV, MuV, and RuV IgG antibodies in sera and to compare the sensitivity to commercial enzyme immunoassays. The IgG antibody levels to MeV and MuV were measured using EIA test kits for a total of 44 healthy serum samples. Of the samples, 6% were seronegative for MeV-specific IgG antibodies and 75% were seronegative for MuV-specific antibodies, showing low correlation of IgG antibody levels between both viruses.
To improve the sensitivity of the POC device, multiple conjugated fluorescent secondary antibodies were tested with different surface chemistries. Signal detection was measured using the pre-developed four-site slide reader. Preliminary data show that Nile Red microspheres provide robust signal detection and should be the secondary antibody of choice when sera are tested for IgG antibodies using the POC platform in future work.

Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens are potential diagnostic biomarkers of HPV-associated OPC (HPV OPC). A custom multiplexed bead array assay was used to detect Abs to HPV16 antigens E1, CE2, NE2, E4, E5, E6, E7, L1, and L2. Following extensive optimization of the assay, these genes were expressed as GST-fusion proteins and captured onto anti-GST magnetic beads. Serum was obtained from 256 OPC patients at the time of diagnosis and from 78 healthy controls. The median fluorescent intensity (MFI) was determined for each antigen and ratios of MFI to control GST-fusion protein were determined for each serum sample. Cutoff values were set as the mean + 3 SD of the MFIs of healthy controls and p-values were calculated using Wilcoxon unpaired and Fisher's exact test. Results of this experiment showed that HPV16 E1, CE2, NE2, E4, E6, and E7 Ab levels were elevated in OPC patients compared to controls (p<0.001), as were Ab levels to L1 (p = 0.013) and L2 (p = 0.023), per Fischer's exact test. Abs to CE2, NE2, E6, and E7 were identified as a potential biomarker panel for early detection of HPV OPC. For the 111 patients with known HPV+ tumors as measured by tumor PCR of E6 and/or E7, this assay had a sensitivity of 90% and specificity of 87% (AUC = 0.96). From these results, we conclude that custom bead array assays can be used to detect HPV16 Abs in patient sera, and we have identified a 4-Ab biomarker panel for the early detection of HPV OPC.

Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in females worldwide, accounting for 23% of all new cancer cases and 14% of all total cancer deaths in 2008. Five tumor-normal pairs of primary breast epithelial cells were treated for infinite proliferation by using a ROCK inhibitor and mouse feeder cells. Methods: Raw paired-end, 100x coverage RNA-Seq data was aligned to the Human Reference Genome Version 19 using BWA and Tophat. Gene differential expression analysis was completed using Cufflinks and Cuffdiff. Interactive Genome Viewer was used for data visualization. Results: 15 genes were found to be down-regulated by at least one log-fold change in 4/5 of tumor samples. 75 genes were found to be down-regulated in 3/5 of our tumor samples by at least one log-fold change. 11 genes were found to be up-regulated in 4/5 of our tumor samples, and 68 genes were identified to be up-regulated in 3/5 of the tumor samples by at least one-fold change. Conclusion: Expression changes in genes such as AZGP1, AGER, ALG11, and S1007 suggest a disruption in the glycosylation pathway. No correlation was found between Cufflink's Her2 gene-expression and DAKO score classification.

The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of the SFK member Lyn as determined by multiplex Luminex assay and verified by immunoprecipitation. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.

Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV strains, only “high-risk” types, may progress to cancer. Serum antibodies to HPV oncoproteins are stable and specific markers that may be able to detect high-grade cervical intraepithelial neoplasia (CIN3). Biomarkers have potential as a rapid, point-of-care HPV screening tool for low resource areas in the way that traditional cytology cannot, and HPV DNA testing is not yet able to.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.