Early diagnosis is one of the key factors to decrease disease-related mortality of advanced prostate cancer. Prostate-specific antigen (PSA) has been utilized as a diagnostic marker for prostate cancer. However, PSA test can cause false positive because enlarged or inflamed prostates also cause increased PSA level. Therefore, new markers are needed to improve the diagnostic accuracy. To discovery novel markers for advanced prostate cancer, we analyzed prostate fluids by mass spectrometry proteomic analysis. We discovered that the regenerating islet-derived protein 3 gamma (Reg3g) is specifically and highly expressed in the prostatic fluids of prostate cancer bearing mice. Reg3g has been shown upregulated in pancreatic cancer and can promote pancreatic cancer progression via STAT3 pathways. Additionally, overexpression of Reg3g induced immunosuppressive tumor microenvironment by recruiting myeloid-derived suppressor cells and enhancing CTLA-4 and PD-1 levels in T cells. Based on these observations, we hypothesize that Reg3g is a novel diagnostic marker and therapeutic target for advanced prostate cancer. We seek to 1) Determine whether overexpression of Reg3g affects prostate tumor cell proliferation and metastasis in vitro. 2) generate a prostate specific Reg3g overexpression transgenic mouse model to investigate whether Reg3g promotes prostate cancer progression and how it affects immune cells infiltration into tumor. Therefore, if we demonstrate that Reg3g expression is closely correlated prostate cancer progression and metastasis, this study will have an immediate impact on diagnosis and treatment of prostate cancer. This proposal addresses two overarching challenges: 1) Develop treatments that improve outcomes for men with lethal prostate cancer and 2) Define the biology of lethal prostate cancer to reduce death.

RNA interference (RNAi) therapy is a high-potential therapeutic solution that utilizes a short-interference RNA (siRNA) molecule to hinder the translation of a target gene. This is especially promising in the application of cancer therapy, as it can be used to suppress mutated oncogenic genes in tumor cells, presenting an alternative to conventional cancer therapy, such as chemotherapy and radiotherapy. However, delivering the siRNA successfully into tumor cells is challenging, as RNA are rapidly degraded in the blood serum. To address this, we developed an alternative siRNA delivery vector using chitosan-PEG coated iron-oxide nanoparticle (IONP:CP). The chemical flexibility of the polymer coating allows conjugation of various molecules, such as siRNA and targeting agents. Red fluorescent protein (RFP) was used as a reporter gene to demonstrate the gene knockdown mediated by the delivery of siRNA with IONP:CP. The siRNA was conjugated onto IONP:CP via disulfide bonds that can be cleaved in a reductive intracellular environment, allowing controlled release of the siRNA. Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) assays and gel electrophoresis were performed to quantify successful siRNA conjugation onto the IONP:CP, while dynamic light scaterring (DLS) and zeta potentials were collected to characterize size and surface charge of the IONP. C6, a rat glioma cell line, transfected with the dsRed plasmid was utilized as our testing platform, and flow cytometry was used to measure fluorescence from the dsRed gene. A significantly low fluorescence levels in the cells would suggest successful siRNA silencing in the cell. We would also compare our data to other conventional methods of siRNA transfection, such as liposomal or polymer based transfection, to investigate the efficiency of our IONP vector.

Cancer is a complex disease, therefore the treatment methods used to address it should be robust enough to effectively combat it. Despite their general success, standard treatments such as chemotherapy or radiation are not suitable for many patients, children in particular, as the associated side effects can be extremely detrimental. Chimeric antigen receptor (CAR) T cell therapy is a solution, as it has shown great promise in combating cancer while overcoming the non-specific nature of traditional therapy methods and minimizing side effects. CAR T cell therapy involves isolating T-cells from a patient's blood and inserting recombinant DNA into them, instructing the cells to target and kill cancer cells. However, there still remains room for improvement, as clinical applications of this therapy method have shown low T cell persistence. This project works to address this issue by introducing a secondary transgene into the system, constitutively activate signal transducer and activator of transcription (CA-STAT). This pro-proliferation molecule, however, may result in T cell senescence when constitutively expressed for extended periods of time. The drastic proliferation may also cause severe side effects in certain patients. Implementing logic gate regulation in the system to tightly regulate the expression of CA-STAT may be the solution. Combinations of an inducible synthetic promoter and drug regulation will be explored to achieve time and location control of transgene expression to minimize its side effect. The synthetic promoter requires CAR activation for expression of the downstream CA-STAT molecule. Fusion of estrogen receptors (ER) to the molecules controls their expression, which is only possible when Tamoxifen binds to the receptor. Efficacy of these systems will be explored by applying them in in vitro and in vivo models. The results will better inform the development of more regulated, personalized and efficient CAR T cell therapy methods for treating cancer in patients.

Skin cancer is the most prevalent cancer in the U.S., with an annual incidence of 5.5 million, exceeding all other cancers combined. Given the extremely high incidence of skin cancer, preventive measures are important. Intriguingly, multiple human epidemiological studies have demonstrated that caffeinated coffee, which is widely consumed in the U.S., is associated with decreased risk of developing skin cancer in a dose-dependent manner. Importantly, decaffeinated coffee had no such preventive effect. To determine the per-cup effect of caffeinated coffee consumption on skin cancer prevention, we performed linear regression analyses of previously published data. Males showed 3.0% reduced risk per cup of caffeinated coffee, whereas females showed 4.4% reduced risk. To determine the average reduced risk among males and females in the U.S. population, we extracted coffee consumption data from the National Health and Nutrition Examination Survey (NHANES) 2015–2016. Subsequently, the average number of cups of caffeinated coffee consumed was combined with the per-cup effect on skin cancer prevention. Among caffeinated coffee drinkers, males and females consumed on average 2.9 and 2.0 cups, respectively. However, both male and female caffeinated coffee drinkers coincided with 9% reduced risk of skin cancer incidence even though coffee consumption and the per-cup effect on risk reduction were different between males and females. The cancer-preventive effects of caffeinated coffee per cup appear to be stronger in females than in males. However, greater coffee consumption in males with weaker per-cup effect on skin cancer prevention leads to the net effect in males being comparable to that in females. For both genders, current levels of caffeinated coffee consumption may be preventing thousands of skin cancers annually.

RNA viruses, such as influenza, are known for their ability to quickly fix mutations. Some of these mutations are selected for because they change the region of the virus targeted by the immune system and thus allow the virus to escape the immune response. This rapid evolution can be detrimental to human health as it limits the effective duration of vaccine-conferred immunity. Rapid evolution in viruses also offers a unique system in which to investigate important basic questions in evolutionary biology, especially given that recent advances in deep sequencing now allow almost real-time observation of virus evolution. Here, we use empirical data from a high-throughput functional assay known as deep mutational scanning to define the null expectation for the evolutionary rate of viruses when subject to only purifying selection. These null models differ from traditional phylogenetic models in that they describe evolutionary constraints on a site-specific basis, offering a major advantage in statistical power. We have implemented a random-effects-likelihood statistical approach to identify sites which deviate from our null models by an unexpectedly high evolutionary rate, i.e., sites under positive selection. We used simulations to evaluate the statistical power and accuracy of our approach. Preliminary results show that our method outperforms other methods for identifying sites under positive selection, e.g., with increased sensitivity. Next, we will apply these methods to viral proteins such as influenza hemagglutinin, HIV envelope glycoprotein, and Zika virus envelope protein. Identifying sites under positive selection on viral proteins could help predict future circulating strains, inform structure-based vaccine design, and help to understand basic evolutionary questions.

Immunological memory prevents reinfection by a pathogen. This protection is accomplished by memory T cells expressing T cell receptors (TCR) specific for previously encountered pathogen-derived peptides (antigens). Conventionally, memory T cells are thought to be inert during novel infections because there is no interaction between their TCRs with their specific antigens. Despite this, we and others have demonstrated that these T cells (here termed “bystanders”) can be activated by inflammatory signals alone and gain cytotoxic effector function in the absence of TCR-antigen interaction. This study aims to determine how inflammation regulates and attenuates bystander responses and how we can leverage these cells therapeutically. Using in vitro cell stimulations, we found that the inhibitory receptor, programmed cell death protein 1 (PD-1), is strongly upregulated by bystanders after exposure to certain inflammatory cytokines. This finding is unique because the current paradigm is that PD-1 expression is caused by TCR stimulation and PD-1 represents a target to manipulate bystander responses. Further, in mouse models of vaccination, we found that bystander-mediated killing can limit vaccine antigen. We believe that interfering with bystander T cell effector functionality could be targeted to improve antigen-specific vaccine responses. Through understanding the mechanisms that dictate bystander function, we may better modulate bystander T cells function during infection, vaccination, and cancer to improve patient outcomes.

The recent discovery of neoantigens has opened a new direction in the investigation and development of therapeutic cancer vaccines. Neoantigens are tumor-specific peptides displayed on the surface of cancerous cells with mutations that distinguish them from the peptides displayed on healthy cells, and they are ideal non-self-targets for cancer therapies. Previously developed therapeutic vaccines utilizing neoantigens have been able to generate cytotoxic T cell responses against tumors, but the hydrophobic nature of these peptides means they are often formulated for delivery in toxic organic solvents. My aim is to develop a neoantigen vaccine platform using a self-assembling protein cage designed by the King Lab in the Institute for Protein Design. I3-01 is an extremely robust, single component icosahedral protein cage. Studies have shown that I3-01 remains assembled in 50% DMSO, allowing neoantigens to be conjugated to the interior of I3-01 in organic solvent, and then the conjugated cage can be dialyzed back into aqueous solution, effectively solubilizing the peptides. I used better characterized test peptides to establish a conjugation protocol and determined the ideal ratio of peptide to monomer. Simultaneously, I expressed a version of I3-01 in mammalian cells which resulted in the protein subunits being glycosylated, and compared its stability and conjugation efficiency to that of the original cage. In the future, multiple peptide sequences could be conjugated to the cage, to give a broader immune response, and other antigens, adjuvants, or ligands can be displayed on the surface of the cage to allow for further targeting of the nanoparticles. Additionally, packaging neoantigens in a protein-based nanoparticle may improve trafficking and delivery of the antigens to lymphocytes in vivo. This extremely stable platform for delivering neoantigens could be a highly customizable cancer therapeutic, elicit potent immune responses, and fight cancer without the use of toxic drugs or radiation.

 Homing endonucleases are highly specific DNA cleaving enzymes that are encoded by mobile genetic elements that allow for the invasion of coding sequences within a host genome. These site-specific nucleases generate double-strand breaks and they can be altered for novel gene targeting. I-Onul, a member of the “LAGLIDADG” class of homing endonucleases, has been engineered to disrupt expression of PD-1 in T cells, leading to the restoration of the effector activity of exhaustive T cells against solid tumor cells. However, the role of enzyme residues that were altered during each stage of the engineering process to the enzyme’s stability, behavior and DNA recognition specificity is unclear. In order to visualize the outcome of sequential engineering steps that led to eventual optimization of the enzyme, a series of selected constructs were expressed and purified. Their thermostability and enzymatic activity were determined and the structures of the five variants I-Onul are being solved by x-ray crystallography to identify how DNA recognition specificity is accomplished. So far, three structures have been solved. Our work emphasizes the importance of understanding the chemistry between DNA and protein interactions to develop better equipped endonucleases for gene targets of interest.