Huntsman Cancer Institute Sessions: Oral

Cancer affects millions of individuals worldwide and remains a leading cause of mortality. A key determinant of cancer severity is its metastatic potential—the ability of malignant cells to spread from their primary site and establish secondary tumors in distant tissues. Exosomes, nano-sized extracellular vesicles containing proteins, lipids, and nucleic acids derived from their parent cells, have emerged as critical mediators in cancer progression. They play essential roles in tumor growth, immune modulation, and the metastatic process, making them a significant focus of current cancer research. We used various methods of assessing the metastatic potential of AGS, A549, and A375 cells including using the xCelligence invasion assay, treatment with the growth arrest-specific protein (GAS6), and a scratch assay. We identified changes in exosome yield in association with increased metastatic potential. Increased exosome concentration was seen with the scratch assay as well as GAS6 treatment. Additionally, we determined that the treatments did not impact the size of the exosomes collected from each condition. These findings highlight the critical role of exosomes in understanding the migrative potential of cancer cells, underscoring their importance as potential biomarkers and therapeutic targets in limiting metastasis.

Genetic studies have greatly advanced our understanding of the molecular and genetic drivers underlying the steps of melanomagenesis. Although activation of the mitogen-activated protein kinase (MAPK) signaling pathway, specifically in the form of a single driver mutation in the oncogenes BRAF or NRAS triggers melanocyte proliferation in over 60% of cases, mutations in the RAS pathway alone are not sufficient for melanoma formation. However, the addition of activating tumorigenic mutations or loss of tumor suppressor functions in the phosphoinositide 3-kinase (PI3K) pathway promotes the escape from oncogene-induced senescence leading to the formation of malignant melanomas. Dysregulation of the PI3K lipid signaling cascade in melanoma is conventionally believed to occur through activation of AKT, yet it has not been shown whether constitutively active AKT can substitute for mutationally activated PI3K or loss of PTEN in mutant BRAF melanomagenesis in the absence of CDKN2A loss. In this study, we investigated the potential of aberrant MAPK signaling through mutational activation of BRAF to cooperate with dysregulated AKT signaling to drive melanoma tumor formation. We utilized an established autochthonous melanoma mouse model to evaluate the ability of constitutively active Akt1 to promote tumor initiation and progression and observed that genetic alterations in both BRAF and Akt1 are sufficient to promote melanomagenesis with an average tumor onset of 130.5 ± 32.5 days and a median overall survival of 178 ± 2.70 days. Future research will be conducted to evaluate tumors from BRAF + AKT versus loss of PTEN and activated PIK3CA-driven melanomas using RNA sequencing and Reverse Phase Protein Array technology to assess differential pathway activation among the various genetic alterations. We have previously established the potential of myrAkt1 to enhance tumor activity and metastasis with additional genetic events such as loss of the tumor suppressors PTEN and CDKN2A. We now demonstrate that not only does Akt1 lead to melanoma progression and promote metastasis, it also plays a role in melanoma initiation. Results from this study highlight the ability of multiple alterations in the PI3K/AKT signaling pathway to influence BRAF mutant melanoma initiation, progression, and metastasis.

Extracellular vesicles (EVs) are a type of nanoparticle that are secreted by many types of cells within organisms and can be derived from various bodily fluids, such as blood, milk, saliva, urine, and more. These EVs have been isolated from raw cow’s milk, due to milk being easily accessible and not triggering an immune response within the body that would counteract the EVs ability to localize to the brain. EVs have shown promise as transportation agents in vivo, due to their innate abilities to localize to a particular place within the body, their selective release kinetics, and their ability to cross through the blood brain barrier (BBB). As a result of these innate abilities, we have been able to encapsulate temozolomide (TMZ), a chemotherapeutic drug used in the treatment of glioblastoma, into EVs. This has been accomplished via electroporation, which causes pores to form in the EV membrane, allowing for the uptake of amphipathic TMZ molecules. To determine the efficacy of these EVs as a transportation system, this project will be focusing on the release kinetics of the TMZ-encapsulated EVs. The release kinetics study of TMZ-encapsulated EVs will occur through the use of dialysis and will focus on the ability of the EVs to properly release their drug cargo into the surrounding solution, which is representative of their ability to release the encapsulated TMZ into glioblastoma cells. This project allows for better understanding of the mechanisms and timeline by which the EVs will deliver chemotherapeutic drugs into target tissues.

Background and Purpose:
An estimated 85,480 adolescents and young adults (AYAs) between the ages of 15 and 39 will be diagnosed with cancer in the United States in 2025, and this number is growing. AYAs from ethnic and racial minority groups often experience a higher symptom burden, which can lead to a lower quality of life and higher mortality rates. The aim of this project is to compare the prevalence and associated characteristics of common cancer-related symptoms and their associated characteristics among Hispanic AYAs with cancer and non-Hispanic, White AYAs with cancer, as well as differences in priority symptoms reported by Hispanic and non-Hispanic White AYAs with cancer. Methods: This project will involve a secondary analysis of patient-reported symptoms from 86 AYAs receiving treatment for cancer. These AYAs participated in a study evaluating the efficacy of a digital health intervention, the Computerized Symptom Capture Tool (C-SCAT), to improve self-efficacy for symptom self-management. AYAs reported the presence, severity, and associated distress of 32 common cancer-related symptoms using the C-SCAT at two scheduled visits for cancer treatment. Data will be analyzed using SPSS.

Results to date and planned analyses:
Data analyses are underway and will be completed by January 2026. There were 86 participants between 15 and 29 years of age (mean 21 years), 47 (55%) of whom were male. Racial/ethnic representation included 19 (22%) Hispanic, 56 (65%) white/non-Hispanic, and 11 (13%) other race/non-Hispanic. The most frequently reported priority symptoms across each of the two treatment-related visits were pain, fatigue, difficulty sleeping, and nausea. Planned analyses include analysis of variance (ANOVA) tests and non-parametric tests to compare differences in the symptoms and their associated characteristics at each of the two clinical visits, based on race and ethnicity.

Conclusion:
By examining the data provided, we will be able to investigate cancer symptoms reported by AYAs of different racial and ethnic groups (specifically non-Hispanic white vs Hispanic) and what their priority symptoms are. These data can guide further investigation into why these differences occur. It is important to address this problem in order to better understand how the healthcare system is set up, and how to change it so that it does not give preference to one racial/ethnic group over another.

The immune system identifies self versus foreign threats by recognizing the expression of surface markers called antigens. To recognize a foreign antigen, antigen-presenting cells (APCs), primarily dendritic cells, absorb the antigens found on the tumor. APCs then deliver the antigens to T cells, which allows them to recognize the foreign substance and destroy it. Tumors escape immune recognition by altering their surface antigens or the behavior of the local immune environment trying to find them. Tumors can reduce or completely eliminate their expression of certain stimulator antigens, increase expression of "self" antigens, or express antigens which decrease immune function. The high rate of antigen reception bombarding the T cells can lead to T-cell exhaustion, wherein T cells can incrementally lose vital functions assisting in tumor destruction to the point of their death. Loss of these key immune effector cells complicates current clinical therapies. With the goal of designing more effective future therapies, MITI labs utilize multicolor flow cytometry to profile prognostic markers in cancers on several immune axes at the same time. Primarily, we are investigating the expression of two sets of immune axes which signify tumor modulation and immune activation respectively. The CD155 (ligand) CD96 (receptor) and PDL1 (ligand) PD1 (receptor) axes are classic regulators of T-cell exhaustion. They utilize the ligand surface antigens to inhibit immune activity while they engage with the tumor cell surface. Each respective ligand is expressed at higher levels than usual for tumor cells, a clear marker we use to identify them in a large population. Additionally, it allows us to analyze exactly what level each marker is expressed to, alongside the response in corresponding immune cells. MHCII and CD80 are both markers on the surface of APCs which help with the process of presenting antigens and activating T cells. Both MHCII and CD80 expression can be used not only to track the location and activity level of APCs, but to test the general level of adaptive immune response throughout several tissues and cancers. All in all, better understanding the quantitative marker expression in immune-modulated tumor environments will inform better prognostic outcomes in future therapy models acting along these axes.

AlphaFold, an artificial intelligence-based protein structure prediction tool, has revolutionized molecular biology by expediting the traditionally lengthy process of protein structure determination. The tool has the ability to accurately predict single protein structures however, it has difficulty predicting protein-protein interactions (PPIs) [1], [2]. In this study, we use the phospho-binding protein 14-3-3ζ as a case study to test AlphaFold’s ability to predict PPIs, while also using AlphaFold to predict novel 14-3-3ζ interactors. 14-3-3ζ interacts with hundreds of proteins at specific phosphorylated Serines or Threonines and thereby modulates essentially every major pathway in the cell [3]. In many cases, 14-3-3ζ PPIs have been molecularly characterized, including identification of the PPI-mediating phosphorylation, but are not included in AlphaFold’s training data. Therefore, they serve as an excellent test case for evaluating AlphaFold’s predictive accuracy for PPIs. Based on our understanding of 14-3-3 PPIs, we developed criteria to assess the accuracy of 14-3-3-interactor models, including whether AlphaFold places the interactor phosphorylation within the 14-3-3 phospho-binding pocket. Using AlphaFold2 (DeepMind, UK) and ChimeraX (UCSD, USA), we found that AlphaFold was able to predict more than half of the 14-3-3 PPIs correctly even in the absence of assigned phosphorylations on the binding partner [4], [5]. This suggests that AlphaFold recognizes structural features required for 14-3-3 binding that we do not yet understand. Our current work focuses on using AlphaFold complemented with wet lab approaches to identify structural features that mediate 14-3-3 binding. To assess AlphaFold’s ability to identify novel 14-3-3ζ interactors, we used AlphaFold2 to screen every kinase in the human genome for interaction with 14-3-3ζ. To validate the docking results from the screening, results were categorized using confidence measures, and statistical significance was determined via Fisher’s exact test (p<0.0001). From this screening, various unreported interactors scored highly. The highest-scoring unreported interactor was MAST3, a dark kinase. An in vitro study was conducted to evaluate whether AlphaFold correctly predicted this interaction. Co-Immunoprecipitation and western blotting suggest that MAST3 is a 14-3-3ζ interactor. Further studies are being conducted through the use of AlphaFold and in vitro testing to determine if AlphaFold can detect MAST3’s point of interaction with 14-3-3ζ. While AlphaFold cannot yet replace laboratory techniques, its predictions provide a valuable starting point for experimental validation. The successful prediction of known protein-protein interactions show that AlphaFold could lead to the discovery of novel 14-3-3ζ interactors as well as provide more information little known about proteins.

Cancer is a leading cause of mortality worldwide, imposing substantial health, societal, and economic burdens. In 2022, approximately 20 million new cases were diagnosed, and 9.7 million people died from the disease. This burden is expected to rise, with global cancer cases projected to reach 35 million by 2050 due to population growth and aging. Most cancers are driven by genetic mutations that disrupt normal cell growth, with roughly 50% involving defects in the Tp53 tumor suppressor pathway. Reactivating the Tp53 pathway has been shown to induce apoptosis in cancer cells and inhibit tumor growth. In this study, we investigated a novel patented gene therapy approach that delivers functional copies of the Tp53 gene to cancer cells. We hypothesized that restoring Tp53 function would trigger programmed cell death and reduce tumor proliferation. Our preliminary in vivo and in vitro studies demonstrate promising results, particularly in triple-negative breast cancer models. These findings support further investigation of Tp53-based therapies as modulators of apoptosis across multiple cancer types.

Background and Purpose:
The United States Healthcare system is founded on the principle of equitable care for all individuals, yet a growing body of literature highlights persistent disparities in health outcomes among racial and ethnic minorities. Pediatric cancer patients are a patient population that experience cancer symptoms in different ways than their adult counterparts. Beyond experience, young patients can struggle to express their symptoms to medical providers, making them difficult to treat. My research aims to describe symptoms and their associated characteristics among Hispanic children involved in active oncology treatment or extended survivorship post treatment. It also aims to compare symptoms and associated characteristics reported by Hispanic children with those reported by White, non-Hispanic children and children of other racial minorities.

Methods:
This descriptive, cross-sectional secondary analysis will use data collected from 95 pediatric oncology patients at Children’s Mercy Hospital in Kansas City, Missouri and Primary Children’s Hospital in Salt Lake City, Utah. Data were collected as part of an instrument development study. Patients screened were between the ages of six and twelve years old (mean 9.1 years, SD=2), with 55% of them being boys. Participants were a mean of 27 months from their initial diagnosis (range 1-109). Of these 95 patients, 19 self-identified as Hispanic/Latino. The study measure was the Memorial Symptom Assessment Scale for 7-12 year olds which assesses the presence, severity, and associated distress of eight common cancer-related symptoms.

Planned Analysis:
Data analyses are underway and due to be completed in January 2026. Planned analyses include comparisons between Hispanic/Latino children (n=19), White/non-Hispanic children (n=60), and children of other racial/ethnic minorities (n=16). Children reported a median of two symptoms (range 0-7). The most frequently reported symptoms were fatigue (n=38, 40%), pain (n=27, 28%), and worry (n=25, 36%). Measures of central tendency will characterize each subgroup. One-way analysis of variance (ANOVA) tests and non-parametric tests will compare differences in prevalence, severity, and associated distress.

Conclusion/Implications:
It is vital to understand if there are groups of patients that have, on average, higher symptoms and associated distress. This study aims to understand possible symptom variation in pediatric patients that are Hispanic in comparison to non-Hispanic, White patients. Due to distress affecting overall patient well-being, minimizing suffering related symptoms should be a priority for medical professionals. The understanding of patient experience is vital to equitable healthcare.

Endometrial cancer (EC) is the most common gynecologic malignancy in the United States, and is also the only cancer type that has seen a decrease in five-year survival rates since 1970. Despite high prevalence and increasing incidence, the field lacks molecularly informed therapies and instead relies heavily on surgical intervention (hysterectomy) for treatment.

Although this field has scarce options for targeted therapies, several important EC oncogenes and tumor suppressors have been identified. The Cancer Genome Atlas identified ARID1A, an important subunit of the ATP-dependent nucleosome remodeling complex BAF, as being mutated in 54% of cases with nonsense truncation events being most prevalent. The high mutational frequency of ARID1A suggests that it is a tumor suppressor. ARID1B, ARID1A’s mutually exclusive paralog, is not frequently mutated in EC, suggesting that ARID1B has no such tumor suppressive role. Despite their relatedness (>50% sequence identity), ARID1A and ARID1B appear to have independent functions that are poorly understood.

To investigate the molecular roles of both ARID1A and ARID1B, I modulated their expression in a human endometrioid endometrial carcinoma cell line. This line lacks ARID1A while constitutively expressing ARID1B, and we engineered it to express ARID1A under a doxycycline-inducible promoter, creating an ARID1A/ARID1B expressing line. To remove ARID1B, I subsequently optimized an siRNA-mediated knockdown system in this cell line. I then used live-cell imaging assays to measure cell proliferation after ARID1A induction and after ARID1B knockdown. I’ve found that ARID1A loss increases cell proliferation, whereas ARID1B knockdown decreases cell proliferation. While current models assume that ARID1A and ARID1B play mostly compensatory functional roles, my results suggest that opposing phenotypic outcomes are caused by ARID1A and ARID1B. My ongoing studies are focused on identifying the protein domains and molecular mechanisms that distinguish the roles of ARID1A and ARID1B in EC.

A C-terminal truncation of the non-receptor tyrosine kinase TNK1 in Hodgkin lymphoma generates a constitutively active form of the kinase that drives tumor growth. (Gu et al. 2010) Our laboratory found that this C-terminal truncation eliminates a critical inhibitory phosphorylation at Ser502 in TNK1 that normally acts as a binding site for 14-3-3𝛇.(Chan et al. 2021) Point mutations in TNK1 that disrupt 14-3-3 binding, including Ser-to-Ala mutations at Ser502, mimic the effect of the C-terminal truncation in generating a constitutively active and oncogenic kinase. This suggests that 14-3-3 binding to TNK1 is a major regulatory mechanism to keep TNK1 activity in check. However, we do not yet understand on a mechanistic level how 14-3-3 binding inhibits TNK1. To determine how 14-3-3 inhibits TNK1, we are working towards solving the 3D structure of the 14-3-3 TNK1 complex via cryoEM, but as a starting point, we are also using AlphaFold to model the TNK1-14-3-3 complex and in turn generate hypotheses we can test in the wet lab. The AlphaFold models suggest that 14-3-3 binding occludes the kinase domain and may prevent TNK1 oligomerization, which is required for kinase activity. In addition, the model suggests that 14-3-3 binding occludes a ubiquitin association (UBA) domain at the C-terminus of TNK1 that is likely important for localizing TNK1 to some of its substrates. In our preliminary wet lab experiments, we have generated in vitro biochemical and cell-based systems to test these hypotheses. Our preliminary data confirm that phosphorylation of one of TNK1’s substrates, STAT1, cannot occur when the TNK1-14-3-3 complex is present. Additionally, other studies have highlighted the loss of TNK1’s ability to bind ubiquitin when the TNK1-14-3-3 complex is formed. These preliminary studies have begun to elucidate aspects of 14-3-3 regulation on TNK1 activity. TNK1 could be forced into a conformational change that prevents activity, or it could be due to the prevention of TNK1 oligomerization. To deduce which of these hypotheses are correct, we plan to perform a radiolabeled ATP assay to directly track if TNK1 can phosphorylate a substrate while bound to 14-3-3. Additionally, we are evaluating if the ubiquitin association domain helps bring TNK1 in proximity with its substrates through confocal microscopy if p62 antibodies and TNK1 colocalize in the presence of substrate. Determining how 14-3-3 regulates TNK1 will provide insight into how oncogenic mutations activate TNK1 and may shed light on how altered TNK1 activity affects other disease states, like IBD.

Citations
Chan, Tsz-Yin, Christina M. Egbert, Julia E. Maxson, Adam Siddiqui, Logan J. Larsen, Kristina Kohler, Eranga Roshan Balasooriya, et al. 2021. “TNK1 Is a Ubiquitin-Binding and 14-3-3-Regulated Kinase That Can Be Targeted to Block Tumor Growth.” Nature Communications 12(1): 5337. doi:10.1038/s41467-021-25622-3. Gu, T.-L., J. Cherry, M. Tucker, J. Wu, C. Reeves, and R. D. Polakiewicz. 2010. “Identification of Activated Tnk1 Kinase in Hodgkin’s Lymphoma.” Leukemia 24(4): 861–65. doi:10.1038/leu.2009.293.

Extracellular vesicles (EVs) are nano-sized messengers released by cells, playing a critical role in intercellular communication throughout the human body. Their small size allows EVs to easily pass through various biological barriers, making them effective vessels for drug transport directly into cells. Extracellular vesicles are not only limited to simply passing through biological tissues, they also are capable of surface decoration with structures such as glycoproteins that can target specific body tissues. These unique characteristics have positioned EVs as desirable candidates for drug delivery, exhibiting not only reliable drug delivery, but target specificity. This sets them apart from various drug delivery candidates such as nanoparticles, which lack a lipid bilayer or micelles which present difficulty with specific tissue targeting. While promising, extracellular vesicles are not devoid of challenges. A major challenge in utilizing EVs for therapeutic purposes lies in the lack of standardized protocols for their long-term storage, which is typically done at lower temperatures. Exosomes are found in biological systems where temperature and pH are tightly regulated. When exposed to new environments, extracellular vesicles are vulnerable to decomposition and decreased stability. To address this issue, a comprehensive stability study investigating the effects of different storage temperatures and thawing techniques on isolated EVs over a 12-month period has been initiated. Various methods used to detect the concentration and quality of extracellular vesicles after being subjected to different storage temperatures and thawing techniques have been used. The goal of this research project is to optimize these extracellular vesicles stability while in storage. This will consequently increase their capacity to deliver drugs to target tissues in an specific and effective manner.

The introduction of cancer immunotherapies, immune checkpoint inhibitors (ICIs) that target inhibitory receptors in immune cells such as CTLA4 and PD-1, have revolutionized treatment options for patients with advanced melanoma. Our research aims to develop a new preclinical model appropriate for the study of irAEs caused by ICI treatment using the non-obese diabetic (NOD) mouse line, as it is genetically predisposed to many autoimmune diseases.

Melanoma is a type of cancer that begins in melanocytes, the pigment-producing cells of our skin. Primary tumors can develop anywhere and can spread to other parts of the body, including distant organs. The most deadly organ for melanoma to metastasize is the brain, with brain metastasis accounting for 54% of melanoma deaths [1]. Despite advancements in treatment, survival after diagnosis for patients with melanoma brain metastases (MBMs) is typically less than 2 years [2]. As such, it is crucial to investigate the mechanisms by which melanoma cells metastasize to the brain, in hopes of finding new potential therapeutic targets.

One intracellular pathway that is often upregulated in metastatic melanoma is the phosphatidylinositol 3’- kinase/protein kinase B (PI3K/AKT) pathway. This pathway regulates cell survival, growth, proliferation, and invasion, and overactivation can promote tumor growth and metastasis. Previous work done in our lab found that activation of AKT1 promoted melanoma metastasis to the brain and lungs and we identified focal adhesion kinase (FAK) as one of its downstream targets. Further, the presence of activated AKT1 changed the expression of integrin subunits 𝛼v, 𝛼5, β1, and β5 [3].

Both FAK and integrins are highly involved in mediating cell adhesion, which plays a crucial role in metastasis. In our mouse models, the expression of activated FAK was sufficient to promote melanoma brain metastasis. Studies have demonstrated that FAK is activated through integrin-mediated signaling and differential expression of integrins, including 𝛼v, 𝛼5, β1, and β5 have been found in MBMs [4][5]. This project further investigates the potential role that integrins play in melanoma brain metastasis.

We hypothesize that overexpression of these integrins promotes melanoma cell migration and invasion in vitro via interactions with FAK. To test this, we utilized Gateway technology to clone integrin subunits 𝛼v, 𝛼5, β1, and β5 into lentiviral vectors containing either GFP or mCherry. These plasmids were transfected into human embryonic kidney cells and the virus-containing media was used to infect Yale University Mouse Melanoma cells (YUMM3.2) known to be non-metastatic in our mouse model. These YUMM3.2 cells were sorted via flow cytometry based on GFP or mCherry expression and surface staining. They were then co-transfected, creating additional cell lines containing combinations of alpha and beta subunits. We will present our analysis of the ability of the YUMM3.2 cells with or without expression of these integrins to migrate and cross the blood-brain barrier via a migration and invasion assay.

References

[1] R. L. Siegel, K. D. Miller, and A. Jemal, “Cancer statistics, 2017,” CA: A Cancer Journal for Clinicians, vol. 67, no. 1, pp. 7–30, Jan. 2017, doi: https://doi.org/10.3322/caac.21387.

[2] S. Sloot et al., “Improved survival of patients with melanoma brain metastases in the era of targeted BRAF and immune checkpoint therapies,” Cancer, vol. 124, no. 2, pp. 297–305, Oct. 2017, doi: https://doi.org/10.1002/cncr.30946.

[3] D. A. Kircher et al., “AKT1E17K Activates Focal Adhesion Kinase and Promotes Melanoma Brain Metastasis,” Molecular Cancer Research, vol. 17, no. 9, pp. 1787–1800, Sep. 2019, doi: https://doi.org/10.1158/1541-7786.mcr-18-1372.

[4] Z. Zhang, J. Li, S. Jiao, G. Han, J. Zhu, and T. Liu, “Functional and clinical characteristics of focal adhesion kinases in cancer progression,” Frontiers in Cell and Developmental Biology, vol. 10, Nov. 2022, doi: https://doi.org/10.3389/fcell.2022.1040311.

[5] R. Huang and E. K. Rofstad, “Integrins as therapeutic targets in the organ-specific metastasis of human malignant melanoma,” Journal of Experimental & Clinical Cancer Research : CR, vol. 37, Apr. 2018, doi: https://doi.org/10.1186/s13046-018-0763-x.

Adrenocortical carcinoma (ACC) is a rare yet deadly cancer of the outer layer of the adrenal gland. The 5-year survival rate is < 40%, and no new treatments have been FDA approved since Mitotane in 1970. Aberrant activation of the Wnt pathway, a key regulator of cell growth and differentiation, correlates strongly with later stages of ACC and poor prognosis. Our lab developed a novel mouse model of ACC by selectively inactivating Znrf3 in the adrenal cortex. ZNRF3 is an E3 ubiquitin ligase that targets Frizzleds (FZDs), the primary receptors of the Wnt pathway, and therefore acts as a Wnt signaling inhibitor. Prior studies discovered that ~20% of ACC patients harbor loss-of-function (LOF) alterations for Znrf3, causing heightened Wnt pathway activity. Using our model, we found that female mice are far more tumor-prone than males, which mimics the increased incidence of ACC in female patients. Tumor protection in males correlates with a significantly reduced expression of Wnt5a, a beta-catenin independent Wnt ligand. Moreover, we found that high WNT5A expression correlates with poor prognosis, higher tumor stages, and suppression of anti-tumor immune pathways in ACC patients. To test if WNT5A indeed promotes ACC, we engineered a Znrf3;Wnt5a double knockout (DKO) mouse model. Using immunohistochemistry to characterize the immune response within adrenal tumors, and ultrasound imaging to track tumor incidence, we saw that DKO females had a far better overall survival and decreased tumor incidence when Wnt5a was inactivated. Importantly, we also observed an increase in myeloid immune cell infiltration, which aligned with the degree of myeloid infiltration in tumor-protected males. These myeloid cells were found to correlate with increased T-cell infiltration in mice and better overall survival in patients, providing evidence that this myeloid response is anti-tumor. These findings supported our hypothesis that inhibition of Wnt5a is tumor protective, however translating these findings to the clinical setting is challenging, as direct inhibition of a secreted factor is rarely effective. Therefore, our ongoing work aims to identify the FZD receptor that interacts with WNT5A to produce this immune response and to test the therapeutic capabilities of inhibiting this receptor in our mouse and human ACC models. This inhibitor could be a powerful tool in combination immunotherapy regimes, which have yet to yield effective responses in ACC patients, by boosting the ability of the immune system to access and regress tumors.

Liver cancer is one of the most aggressive and lethal forms of cancer, with current effective treatments being accompanied by horrible side effects. Hepatocellular carcinoma (HCC) is a particular form of cancer that originates in liver cells. HCC has a 5-year survival rate of less than 20%, which decreases to 3% if the cancer has metastasized (Singal et al., 2023). In 2020, liver cancer was found to be the 6th most common cancer and 2nd most lethal cancer (Tümen et al., 2022). The rate of death for patients with liver cancer has doubled, while the rate of diagnosis has tripled since the 1980s (American Cancer Society, n.d.). Short interfering RNAs (siRNAs) have been used as a natural mechanism for regulating gene expression within a cell. When siRNA is introduced into a cell, a protein complex called RISC (RNA-induced silencing complex) leads it to the matching mRNA strands and cuts them (Dana et al., 2017). Since the destruction of mRNA is very specific, it allows for an effective method of preventing the production of cancer-causing proteins. This method also offers the opportunity to target and kill cancer cells while limiting damage to the healthy surrounding liver cells. Cancer genes, such as astrocyte elevated gene-1 (AEG-1/MTDH), are overexpressed in liver cancer, causing an overproduction of proteins, leading to uncontrolled cell growth. AEG-1 is a gene that in normal circumstances can be found to play roles in metabolism, stress, fertility, and inflammation. AEG-1 is commonly highly upregulated in HCC and has been found to evade apoptosis and play an important role in metastasis (Davis et al., 2025). Validation and cell death (MTS) assay experiments for the AEG-1 oncogene have been ongoing in our lab in two HCC cell lines- Hep3B and PP5. Validation experiments (Figure 1) have shown the AEG-1 siRNA reduces expression of the target gene in both PP5 and Hep3B liver cancer cells. Additionally, the results of our preliminary studies have demonstrated that treatment of sorafenib combined with AEG-1 drastically reduces the IC50 (the amount of sorafenib needed to kill half of the cancer cells) in the Hep3B cell line (see Figure 2). We plan to begin experimentation with N-acetylgalactosamine conjugated with siRNA (NGalAc), which is a method of delivering siRNA treatment specifically to liver cells that has been used successfully in several FDA-approved therapies. If these experiments are successful, we will start liver cancer mouse model experiments.

Despite advancements in treatment, the five-year survival rate for Stage IV melanoma remains low, around 30%, highlighting the urgent need for new therapies. Two key signaling pathways, RAF,MEK,ERK (MAPK) and PI3K,AKT, are often co-activated in melanoma and play a central role in its initiation and progression. About 50% of melanomas have a BRAF mutation, but many patients develop resistance to therapies targeting this pathway. Additionally, disruptions in the PI3K>AKT pathway, such as loss of the tumor suppressor PTEN or activation of PI3K or AKT, are common in BRAF-mutant melanomas. Despite promising preclinical studies, no PI3K or AKT inhibitors have been approved for advanced melanoma treatment. The serine/threonine kinase SGK1 (serum and glucocorticoid-regulated kinase 1) plays a pivotal role in cellular processes such as survival, proliferation, and migration, and has been implicated in various cancers, including melanoma. Our previous work has shown that AKT was inhibited, SGK1 was upregulated, potentially rescuing cell survival and acting as an inherent resistance mechanism for AKT inhibition. SGK1 was also found to be higher in primary melanoma tumors compared to normal skin lesions. In vivo experiments in a mouse melanoma model demonstrate that overexpressing SGK1 promotes tumor growth and reduces survival, and these experiments are currently ongoing to examine the impact on metastasis in this mice. These findings position SGK1 as a promising therapeutic target for melanoma, particularly in combination with existing treatment strategies. This body of work underscores the importance of further investigating SGK1’s molecular mechanisms and its potential to improve melanoma treatment outcomes.

For the last decade, Utah has had one of the highest incidence rates of melanoma, a cancer that develops in the pigment-producing cells of our skin, known as melanocytes. Melanoma is the deadliest form of skin cancer due to its propensity to metastasize to distant organs, especially the brain. In many advanced melanoma patients, the leading cause of death is brain metastasis. While the standard of care, immunotherapy, targeted therapy, and radiotherapy work for a small percentage of patients, more effective therapies for patients with melanoma brain metastasis are needed to improve patient outcomes. However, to develop novel treatment strategies, we must gain a deeper understanding of the biological mechanisms that drive melanoma progression and metastasis. One of the most frequently overactivated cell signaling pathways in melanoma brain metastases is the PI3K/AKT pathway. The PI3K/AKT pathway regulates numerous cellular processes, including cell migration and proliferation, apoptosis (controlled cell death), gene transcription, metabolism, and angiogenesis. Hyperactivation of the PI3K/AKT pathway can occur through various mechanisms, the most common of which involves the loss or deletion of the tumor suppressor gene PTEN. PTEN is the second most commonly mutated tumor suppressor gene in cancer, and it encodes a phosphatase with dual activity against lipids and proteins. Phosphatases are a group of enzymes that remove phosphate groups, negatively charged molecules, from other proteins, altering cell signaling. In this case, PTEN acts to suppress the PI3K pathway by dephosphorylating various proteins and lipids, thereby preventing signaling through key proteins like AKT that drive tumor metastasis. This project aimed to examine how melanoma metastasis is affected when PTEN loses its ability to perform lipid, protein, or non-enzymatic functions. We utilized in vivo mouse models to introduce mutant forms of PTEN encoding a form of the protein that only exhibited lipid or protein phosphatase activities. We found that loss of PTEN lipid phosphatase activity resulted in metastasis to the brain, liver, and lungs, while the presence of lipid phosphatase function completely inhibited melanoma metastasis. Additionally, the addition of FAK with an N-terminal deletion of its FERM domain, which facilitates interactions with lipids at the cell membrane, restored the metastatic phenotype in cells that retained PTEN lipid phosphatase activity. This data demonstrates that FAK functions downstream of PTEN lipid phosphatase signaling to promote metastasis to the brain and other organs, providing valuable insights into the mechanisms by which FAK promotes metastasis in melanoma.

Synovial sarcoma is a soft tissue cancer that is found in the muscle surrounding joints and bones. It is frequently diagnosed in adolescents, and it has very high rate of metastasis with 50-70% of cases metastasizing. Pursuing new treatments for synovial sarcoma that can treat cases with or without metastasis with high specificity has led to our development of a gold-based antibody drug conjugate (ADC) targeting an overexpressed receptor (OSMR receptor) on synovial sarcoma cancer cells. When the gold nanorods conjugated to the anti-OSMR antibody bound to synovial sarcoma cancer cells are excited with near infrared laser light, the resulting thermal energy given off results in thermal ablation of the cancer cells themselves. In analyzing the effectiveness of the laser light reaching the whole of the tumor to cause sufficient thermal ablation, measuring the scattering function, intensity, and area covered by the scattering events due to the light-gold interaction is essential for quantifying how large of an area on the tumor can be targeted with sufficient scattering intensity of the laser light to promote thermal ablation. Here we present the findings of measuring the scattering function, intensity, and area of the near infrared laser light-gold nanorod interaction. This was done by recreating the tumor and normal tissue environment in tissue mimicking agar phantoms with embedded ex vivo synovial sarcoma tumors with the ADC added. To corroborate our findings, computational Monte Carlo simulations were designed and implemented to simulate the physical tissue phantom models and predict the scattering function, intensity, and area values for comparison. It was found from the tissue phantom model that the scatter function and intensity was represented by an exponential decay function of y=247.4e^-0.126x with an area of 10 mm^2 from the light entry point. The Monte Carlo simulation came out similarly with an exponential decay function of scattering intensity of y=389.21e^-0.112x with an area of 10 mm^2 from the light entry point.

The sorting and assembly machinery component 50 (SAM50) is a mitochondrial outer membrane protein essential for maintaining mitochondrial structure, protein import, and inter-organelle communication. Mitochondrial dysfunction is increasingly recognized as a central mechanism in both neurodevelopmental disorders and neurodegenerative diseases such as Parkinson’s disease (PD). However, the specific role of SAM50 in vertebrate neurodevelopment and neuronal maintenance remains poorly understood. This study aims to investigate the functional role of SAM50 in neurodevelopment and PD-related pathways using the zebrafish (Danio rerio) model. Leveraging the advantages of zebrafish genetics and live imaging, we will use CRISPR/Cas9 gene editing to knock out samm50 and its paralogue, samm50l. Phenotypic analyses will include assessments of neural cell markers, reactive oxygen species, mitochondrial morphology, mitochondrial protein transportation, and stress pathway activation. This in vivo approach will provide new insights into the conserved and divergent roles of SAM50 in mitochondrial function and their potential contribution to neurological disorders.

Chimeric antigen receptor (CAR) T cell therapy is an immunotherapy currently approved for the treatment of B cell-mediated cancers. CD19 CAR T cell therapy uses T cells transduced with an anti-CD19 binding domain that binds to and eliminates cells expressing CD19, an antigen found on all B cells, including the cancer. While most treatments are effective, a current limitation is the exhaustion of the CAR T cells as they are chronically stimulated by the CD19 antigen, leading to decreased proliferation, decreased cytotoxicity, and increased expression of surface exhaustion markers. Though the phenomenon of CD19 CAR T cell exhaustion is well-known, the signaling pathways that lead to it are not, thereby limiting our understanding of how to interrupt and prevent its occurrence. Therefore, we aim to gain a greater understanding of the CAR T cell exhaustion pathway by using phospho-proteomics to measure internal signaling pathways of CAR T cells during their basal, effector, and exhausted stages. Human T cells were isolated from blood donors and transduced with the CD19 CAR. The CAR T cells were coincubated with CD19-expressing cancer B cells in a 1:1 ratio, measured as Day 0. Beginning Day 2, after the initial B cells were eliminated, a 4:1 ratio of B cell to CAR T cell ratio was added and replenished every two days. If the 4:1 threshold was breached, CAR T cells were determined to be exhausted. CAR T cell exhaustion occurred after 8 days, and CAR T cells were collected for phospho-proteomics in the basal, effector, and exhaustion stages (Days 0, 2, and 8). Expression of exhaustion markers on CAR T cells was significantly larger than expression on untransduced T cells. Markers LAG-3 and TIM-3 generally increased, but PD-1 generally decreased, due to its initial high expression during the effector stage. The percentage of both AnnexinV+ and dead CAR T cells increased as CAR T cells progressed towards exhaustion. Phospho-proteomic analysis provides insights into key pathways that can be interrupted to prevent CAR T cell exhaustion.

Properly functioning tyrosine kinases are key regulators of cell proliferation. However, constitutive activation of tyrosine kinases leads to overactive downstream signaling pathways and uncontrolled cell growth, resulting in tumor formation. One mechanism of constitutive tyrosine kinase activation occurs through gene fusions, in which a non-kinase gene is attached through chromosomal translocation to the 5’ end of a kinase gene, producing a chimeric oncogenic fusion protein in which the C-terminal end includes a tyrosine kinase domain and the N-terminal end contains a non-kinase partner domain. The non-kinase, N-terminal portion of the fusion can come from many different genes and confer a variety of oligomerization and other functional domains to the kinase. Emerging evidence suggests different N-terminal partners may affect tumor biology in clinically important ways through mechanisms that are not well understood. Our project addresses this gap by investigating how these different N-terminal fusion partners differentially affect 1) the subcellular localization, substrate preferences, and drug sensitivity of their kinase partners; and 2) their tumor aggressiveness in vivo.

Using the Ba/F3 cell system, we generated isogenic cell lines transformed by a panel of kinase fusions with varying N-terminal partners. These fusion-expressing cells no longer need the growth factor IL-3 to survive, indicating that the fusions are truly oncogenic and are driving cell growth/division. We also confirmed that our cells are expressing comparable amounts of fusion protein in relation to human patient-derived cell lines. Additionally, we’ve seen that STAT5 has differing phosphorylation levels between fusions with the same kinase domain but different N-terminal partners, suggesting that the N-terminal partners affect the downstream signaling pathway regardless of the kinase.

Going forward, we will use a mouse model to test tumor aggressiveness and biological effects in vivo, confocal imaging to determine where in the cell fusions localize, and phosphoproteomics to map differences in substrate preferences conferred by the N-terminal partners. The ultimate goal of this research is to improve care for patients with tyrosine kinase fusion-driven cancers. If we can more specifically identify how the N-terminal partners are changing kinase biology and cancer phenotypes, avenues for more personalized treatment will become available.

Blood liquid biopsies are used to detect, diagnose, and monitor cancer through the analysis of key biomarkers such as exosomes, T-cells, and cell-free DNA (cfDNA), which aid in the indication of tumor presence, profiling, and immune activity. An ongoing challenge has been the development of long-term storage methods that maintain the integrity of all three biomarker classes for downstream analysis. In this study, we established and evaluated a standardized workflow in which whole blood was drawn and separated into plasma and peripheral blood mononuclear cells (PBMCs) and subsequently cryopreserved for 37 days. We hypothesized that our integrated liquid biopsy storage method—combining Streck and SepMate tubes with optimized cryofreezing and isolation procedures—would preserve exosomes, T-cells, and cfDNA suitable for downstream analysis. Our results demonstrate that our unique, comprehensive approach to cryopreserving plasma and PBMCs separately preserves cfDNA yield and T-cell viability, while exosome results warrant further research. This approach enables reliable downstream applications across all three biomarker classes.

Gastric cancer shows different tumor immune microenvironment (TIMEs), which influence how tumors might respond to immunotherapy. To better identify these immune states, this study combined isolation of human peripheral blood mononuclear cells (PBMCs) and CD8+ T cells with analysis of extracellular vesicles (EVs) from mouse gastric cancer models representing “hot” (YTN3) and “cold” (YTN16) tumors. PBMCs were isolated through density gradient centrifugation, identified by flow cytometry and ELISA. EVs were collected from plasma and tumor tissue of mice to compare immune-associated protein levels across tumor types. Results showed successful identification of CD8+ T cells in PBMCs and higher CD8+ protein levels in YTN3 tissues compared to YTN16, consistent with greater immune response in hot tumors.

Background:
Antibody-drug conjugates rely on the specificity of antibodies to selectively deliver a cytotoxic payload to cancer cells expressing unique antigens. We aim to design an application with separate components that by themselves are harmless, but when combined result in thermal toxicity to the tumor cells. Gold nanoparticles are innocuous and exhibit localized surface plasmon resonance (LSPR), allowing them to absorb photons at certain wavelengths, causing excitation and the release of heat. In this project the gold nanorods were conjugated to an anti-Oncostatin M Receptor (OSMR) antibody to target OSMR-expressing cancer cells. Thermotoxicity is induced in the cells where the conjugate resides by near IR laser activation of gold nanoparticles.

Methods:
Anti-OSMR antibodies were conjugated with a fluorophore and injected into an in vivo model measuring the antibody’s ability to bind the OSMR receptors. The antibodies were then modified, conjugated with a fluorophore, and introduced to OSMR expressing cell lines in vitro to test the binding ability of the conjugated antibodies post modification. Then, gold nanoparticles were synthesized by a seed growth method. To functionalize the nanorods and increase biocompatibility, the toxic capping agent CTAB coating was replaced with m-PEG-SH via sonication followed by purification using a filtration dialysis cassette. The gold nanorods were imaged using a transmission electron microscope (TEM) to assess size measurements and analysed by UV-VIS spectroscopy to assess the absorbance. The cell viability was tested at varying concentrations of both the CTAB and PEG gold nanorods. The rods were then conjugated with the anti-OSMR antibody, and a UV-VIS was taken to assess the effect on nanorod absorbance.

Results:
The gold nanorods were synthesized and modified successfully. The average length and width of the gold nanorods premodification were 21.1 nm and 6.5 nm respectively, and 16.5 nm and 7.6 nm after substituting the CTAB with PEG. The change in dimensions can be attributed to the change in coating from CTAB to PEG. The cell viability test showed a clear difference in toxicity levels between CTAB and PEG, with the PEG not affecting cell viability significantly at lower concentrations. The spectra observed after running the UV-VIS on the conjugated antibody suggest successful conjugation.

Conclusion:
The successful conjugation of gold nanorods to the anti-OSMR antibody suggests that using a gold-based ADC approach to target OSMR-expressing cancer cells is feasible. The proposed laser and gold nanorod induced thermotoxicity would allow temporal control of this ADC in future testing.

Cluster of differentiation 5 (CD5) and cluster of differentiation 6 (CD6) proteins are two proteins with similar shape and structure that are coreceptors with the T Cell Receptor (TCR). The TCR binds to the antigens that activate T cells. Previous research has shown that removal of the CD5 protein causes a significant increase in basal and maximal respiration of naive helper T cells. The similar protein CD6’s expression correlates with higher survival rates in certain cancers, but is also shown to be more heavily expressed in certain immune disorders such as psoriasis or multiple sclerosis. If the metabolic rate changes when CD6 is knocked out in the same way that it did when CD5 was knocked out, the higher basal metabolic rate would mean that CD6 knockout (CD6KO) T cells may be able to outperform cancers in the tumor microenvironment as they will be more effective at generating ATP. Increased maximal respiration could also increase the T cells ability to handle metabolic stress in the body. Because of the possible positive and negative influences of the CD6 protein, we want to understand more how CD6 affects the metabolism of the cell. We used the Seahorse machine to run a metabolic assay on CD6KO male mice. This assay measures the oxygen consumption of the T cell while inserting drugs at different points to assess the cellular respiration rate of the T cells. During these experiments we used CD8 T cells. CD8 T cells, also known as killer T cells, are part of the adaptive immune system and are vital for eliminating pathogens that enter the body. The results from the seahorse assay on CD8 T cells of CD6KO mice showed CD8 T cells had higher basal metabolic and higher maximal metabolic respiration compared to the control. Further research is needed to fully understand the effect these higher metabolic rates will have on the function of the T cell and how the absence of CD6 affects mitochondrial structure and metabolite levels.

Synovial sarcoma is a rare and aggressive soft-tissue malignancy that most often affects adolescents and young adults. Despite progress in cancer research, this tumor type continues to have limited targeted treatment options and a poor prognosis for patients with advanced disease. As a result, there is a critical need to identify new molecular pathways that can be exploited for therapeutic intervention. Recent studies have shown that sphingomyelin, a membrane-associated sphingolipid involved in cell growth, migration, and metastasis, is upregulated in synovial sarcoma. This upregulation promotes tumor progression by altering lipid signaling and disrupting the balance between cell survival and apoptosis.

Jaspine B, a natural compound derived from marine sponges, has emerged as a potential therapeutic agent due to its ability to inhibit sphingomyelin synthase (SMS), the enzyme responsible for converting ceramide into sphingomyelin. By blocking this enzyme, jaspine B increases intracellular ceramide levels, which promotes apoptosis and suppresses tumor growth. Ceramide is a critical bioactive lipid that regulates cell stress responses, differentiation, and programmed cell death, making it an important target for cancer treatment.

In this study, we examined the effects of jaspine B on sphingomyelin synthase inhibition and cell proliferation in synovial sarcoma cell lines. We also investigated its interaction with spisulosine, a clam-derived inhibitor of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo ceramide synthesis. Both compounds individually reduced cell viability; however, their combination produced a synergistic effect, leading to significantly greater levels of apoptosis compared to either drug alone. These findings indicate that simultaneous inhibition of sphingomyelin synthase and serine palmitoyltransferase may represent a promising and innovative therapeutic strategy for synovial sarcoma and potentially other malignancies driven by elevated sphingomyelin levels and dysregulated sphingolipid metabolism.

Exosomes are extracellular vesicles critical for intercellular communication. This study serves as a step toward tracking the effects on exosome release and contents caused by various conditions in the tumor environment, specifically looking at how starvation affects the ratio of exosomes produced per cell.

Exosomes are extracellular vesicles critical for intercellular communication and implicated in cancer and immune responses. The mechanism and reasoning for the release of exosomes is still a novel idea and something to which the details are widely unknown. This study serves as the first step to tracking the effects on exosome release and contents caused by various conditions that may realistically be placed on the tumor environment. We grew various cell lines involved in the structure of gastric cancer, as well as the body’s immune response to cancer, under conditions of FBS serum starvation and measured cell count as well as exosomes released over a 24hr period. These cell lines were then harvested for both the cells and exosomes produced, and isolated using various isolation techniques. Our findings indicate that there is a statistically significant difference not only among cell and exosome count, but also among the exosome to cell ratio measured across the five cell lines at three different levels of serum starvation. Particularly interesting results included the resilience of the JURKAT cell line as well as an increase in AGS exosome production as starvation levels increased. These results represent progress towards the possibility of having a greater understanding of the happenings of the tumor environment through a simple blood draw. Further research has now begun to address other gastrointestinal cancer related conditions such as hypoxia and chemotherapy. We are also working to analyze the protein components of the exosomes that have been collected to give a greater look into the cell signaling involved.

Background:
Folic acid is an essential vitamin involved in DNA synthesis and repair, processes that are often dysregulated in cancer cells. Its overexpression in tumor tissues via folate receptors has led to the development of folic acid-linked nano-micelles as targeted drug delivery systems.

Aims/Purpose:
This project investigates the potential of folic acid analogs to improve receptor binding efficiency and specificity in nano-micelle–mediated chemotherapy delivery.

Methods:
Computational modeling will be conducted using Schrödinger Maestro to perform molecular docking studies between folate receptor proteins and proposed folic acid analogs. Binding affinities, interaction energies, and docking conformations will be analyzed to identify candidates with superior receptor engagement compared to native folic acid.

Results:
Preliminary modeling data are expected to reveal analogs demonstrating enhanced docking stability and binding strength, indicating improved potential for targeted drug delivery.

Conclusion:
By identifying folic acid analogs with optimized receptor affinity, this study aims to inform future nano-micelle design for more efficient and selective cancer therapeutics.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Despite advances in surgery and systemic therapy, patient outcomes remain poor, and the current standard treatment (sorafenib) provides only modest survival benefits with significant side effects. This project investigates the therapeutic potential of small interfering RNA (siRNA) targeting Ubiquitin-Specific Protease 11 (USP11), a deubiquitinase that regulates the cell cycle, DNA repair, and oncogenic signaling. USP11 is upregulated in multiple HCC cell lines and contributes to tumor growth and metastasis; therefore, its silencing may enhance the response to chemotherapy. To evaluate this hypothesis, USP11-targeting siRNA was transfected into PLC/PRF/5 (PP5), Hep3B, and C3A HCC cell lines. Quantitative RT-PCR confirmed USP11 knockdown to less than 10% of untreated controls. MTS assays demonstrated that USP11 silencing lowered the ICâ‚…â‚€ of sorafenib in both PP5 and Hep3B cells, indicating increased drug sensitivity. Most recently, GalNAc-conjugated USP11 siRNA achieved effective knockdown in PP5 cells without lipid-based transfection reagents, validating receptor-mediated delivery as a less cytotoxic and more consistent approach. Future work will expand GalNAc-siRNA testing to additional HCC cell lines and assess combination therapy with sorafenib. In collaboration with the Parkman Lab, in vivo studies will evaluate therapeutic efficacy in mouse xenograft models. Our research aims to establish USP11 as a viable therapeutic target and advance siRNA-based strategies toward safer, more effective treatments for liver cancer.

Focused Ultrasound (FUS) is a noninvasive therapy for abdominal conditions such as kidney stones or cancerous tumors that focuses high amounts of energy in a small area to ablate tissue or objects. FUS treatments use Magnetic Resonance Imaging (MRI) to monitor the surgery, which sends a Radio Frequency (RF) pulse at tissue protons, then generates images based on the energy they emit as the protons’ spins return to equilibrium. The amount of time it takes to return to equilibrium is measured in segments labeled as T1, T2, and T2* which are crucial for calibrating the MRI to effectively generate images. However, when FUS surgery is used, abdominal adipose (fat) tissue absorbs much of the acoustic energy, causing it to heat up and change its T1, T2, and T2* times, making quality MRI challenging. Without accurate MRI, the treatment becomes ineffective and unsafe. This study aims to describe how the T1, T2 and T2* properties of adipose tissue correspond to its temperature. The setup for the experiment will center around a water bath with an adipose sample placed in an MRI scanner and connected to a water heater in the control room. Using fiberoptic probes to monitor their temperatures and pumps to transfer the heated water between baths, the tissue sample will be heated in 5°C increments. Once the water temperature is equivalent to the temperature throughout the adipose sample, measurements will be taken of T1, T2, and T2*. These times will then be plotted against the adipose tissue temperature to analyze correlations. Previous studies have already found a strong linear correlation of T1 properties to adipose tissue temperature, and similar results are expected regarding T2 and T2* times. These correlations, along with comparisons between each time’s temperature dependence, will be presented in the study. With this information, as FUS heats adipose tissue, the MRI could be used to monitor the fat temperature indirectly through real-time measurements of these MR properties. This can enable safer and more effective treatments even as tissue temperature increases.

This projects evaluates if short-interfering RNA (siRNA) targeting the ECT2 oncogene can increase the vulnerability of hepatocellular carcinoma (HCC) cells to sorafenib (FDA-approved chemotherapy for liver cancer), thereby reducing the effective dose of the drug required for treatment. HCC is one of the most common and deadly liver cancers with only a 22% 5-year survival rate (Calderon-Martinez et al., 2023). Not only are the survival rates dismal, but chemotherapy treatment results in many side effects. Lowering the effective concentration of sorafenib required for treatment could greatly improve patient outcomes. siRNA provides a highly-specific mechanism for silencing the expression of unwanted genes by targeting only the mRNA it is programmed to bind to (Dana et al., 2017). ECT2 is an oncogene that is commonly overexpressed in liver cancers, causing tumor growth by inhibiting natural cellular processes like apoptosis and increasing cell proliferation (Xu et al., 2021). PP5 cells, a commonly studied liver cancer cell line, were grown and seeded into two 96-well plates. One of these plates was treated with ECT2 siRNA and one was left as a control. Then, a series of chemotherapy dilutions were used to treat individual columns in the plate. An MTS Assay was used to determine the concentrations of viable cells left in each well. Initial data shows that treatment of cells with combined sorafenib and siRNA significantly reduces the IC50 compared to treatment with sorafenib alone, indicating improved drug effectiveness at lower concentrations. Future experiments will test the treatment across multiple HCC cell lines to determine consistency of the treatment in a variety of genetic environments.

Calderon-Martinez, E., Landazuri-Navas, S., Vilchez, E., Cantu-Hernandez, R., Mosquera-Moscoso, J., Encalada, S., Al Lami, Z., Zevallos-Delgado, C., & Cinicola, J. (2023). Prognostic Scores and Survival Rates by Etiology of Hepatocellular Carcinoma: A Review. Journal of clinical medicine research, 15(4), 200–207. https://doi.org/10.14740/jocmr4902

Dana, H., Chalbatani, G. M., Mahmoodzadeh, H., Karimloo, R., Rezaiean, O., Moradzadeh, A., Mehmandoost, N., Moazzen, F., Mazraeh, A., Marmari, V., Ebrahimi, M., Rashno, M. M., Abadi, S. J., & Gharagouzlo, E. (2017). Molecular mechanisms and biological functions of siRNA. International Journal of Biomedical Science: IJBS, 13(2), 48–57.

Xu, D., Wang, Y., Wu, J., Zhang, Z., Chen, J., Xie, M., Tang, R., Chen, C., Chen, L., Lin, S., Luo, X., & Zheng, J. (2021). ECT2 overexpression promotes the polarization of tumor-associated macrophages in hepatocellular carcinoma via the ECT2/PLK1/PTEN pathway. Cell Death & Disease, 12(2), 162. https://doi.org/10.1038/s41419-021-03450-z

Melanoma is an aggressive skin cancer characterized by high metastatic potential and frequent resistance to monotherapies targeting the MAPK pathway. Although BRAF inhibitors such as dabrafenib have improved outcomes in patients harboring BRAF V600E mutations, resistance often develops through compensatory signaling and metabolic adaptation. Pterostilbene, a natural polyphenolic compound structurally similar to resveratrol, has demonstrated anti-proliferative and pro-apoptotic effects in several cancer cell lines through modulation of oxidative stress and cell signaling pathways. The present study investigates whether dabrafenib and pterostilbene act synergistically to inhibit melanoma cell growth in the human HT-144 melanoma cell line.

HT-144 cells were cultured in McCoy’s 5A medium supplemented with 2% fetal bovine serum (FBS) and incubated with increasing concentrations of either dabrafenib or pterostilbene for 48 hours. Cell viability was assessed using a presto blue assay, and half-maximal inhibitory concentration (IC₅₀) values were determined for each compound. Dabrafenib exhibited an IC₅₀ of approximately 3 μM, whereas pterostilbene displayed an IC₅₀ of approximately 45 μM under identical conditions. These findings indicate that both compounds exert dose-dependent cytotoxic effects on HT-144 melanoma cells, with dabrafenib demonstrating substantially greater potency. The next phase of the experiment will evaluate potential synergistic interactions between the two agents. Cells will be treated using a 5×5 concentration matrix comprising combinations of 0.5×, 1×, 1.5×, 2×, and 4× the respective IC₅₀ values for each compound. The resulting viability data will be analyzed using a combination index (CI) analysis to determine whether the interaction is synergistic, additive, or antagonistic.

If synergistic effects are confirmed, this combination could represent a promising strategy for reducing effective therapeutic doses and delaying the onset of drug resistance in melanoma treatment. Overall, this study seeks to provide mechanistic insight into the potential co-administration of a targeted kinase inhibitor with a naturally derived compound to enhance melanoma cytotoxicity and improve therapeutic efficacy.

Chimeric antigen receptor (CAR)-T cells are genetically engineered T cells that target surface antigens on cancer cells, showing remarkable effectiveness in treating blood cancers. However, CAR-T cells are currently not effective in treating solid tumors, as the tumor microenvironment poses physical and metabolic barriers to effective CAR-T cell function. A notable strategy for increasing CAR-T cell cytotoxicity in a tumor microenvironment is knocking out or overexpressing some genes of the T cell. We hypothesize that CD6 is a T cell surface protein, which is genetically and structurally similar to the CD5. Both CD5 and CD6 have been shown to have a negative regulatory effect on T cell activation. Removal of CD5 increases the antitumor activity of CAR-T cells against solid tumors (Patel et al., 2024).

We hypothesize that knocking out CD6 will similarly increase the cytotoxic effect of CAR-T cells against tumors. Through a cytotoxicity assay, we are comparing the efficacy of CD6-knockout CD19 murine CAR-T cells with wild type CD19 murine CAR-T cells against tumors. A preliminary assay supported our hypothesis. Future results could yield not only improved CAR-T cell therapy for hematological cancers, but also a potential application in the treatment of solid tumors.

The introduction of cancer immunotherapies, immune checkpoint inhibitors (ICIs) that target inhibitory receptors in immune cells such as CTLA4 and PD-1, have revolutionized treatment options for patients with advanced melanoma, now serving as an effective front-line therapy for patients, and leading to greatly increased survival rates. These immunotherapies enhance the immune system’s ability to locate and eliminate cancerous cells, providing durable anti-tumor immunity. However, ICIs can trigger side effects, termed immune-related adverse effects (irAEs), where autoimmune and autoinflammatory immune responses damage host tissues, impeding physiological function. Severe irAEs can potentially lead to treatment discontinuation, hospitalization or even patient mortality. Immunosuppressive therapies can help mitigate irAEs by dampening the immune response, but may also diminish ICI efficacy, as patients who develop irAEs often exhibit stronger anti-tumor responses. Current mouse models of melanoma are largely resistant to autoimmunity and the development of irAEs following ICI treatment, limiting preclinical studies of mechanisms underlying these toxicities that occur in the clinical setting.

Our research aims to develop a new preclinical model appropriate for the study of irAEs caused by ICI treatment. The proposed model will utilize the non-obese diabetic (NOD) mouse line, as it is genetically predisposed to many autoimmune diseases and develops a broad spectrum of irAEs in response to ICIs, resembling clinical conditions observed in patients. Establishing de novo melanomas that can be initiated in the NOD background allows for direct study of the mechanisms linking tumor immunity and irAEs, in addition to providing a melanoma model to test different combinations of ICIs and targeted therapies. To achieve this, melanoma risk genes will be introduced using the tamoxifen-inducible skin-specific TyrCreER. Specifically, the oncogenic drivers NrasQ61R, CDKN2Afl/fl, and BRAFV600E will be introduced into the NOD background. We hypothesize that inducing melanoma into autoimmune-prone NOD mice will generate a clinically relevant model to test whether targeted or immune-based therapies, used in the setting of melanoma, can enhance antitumor immunity without augmenting the severity and frequency of irAE.

In the United States, ovarian cancer accounts for approximately 2.1% of all cancer deaths, with more deaths than any other cancer of the reproductive system in women. According to the National Cancer Institute, approximately 1.1% of women will be diagnosed with ovarian cancer during their lifetime. After diagnosis, all patients with epithelial ovarian cancer (EOC) are recommended for genetic counseling as part of their clinical workup per National Comprehensive Cancer Network (NCCN) guidelines. Receiving genetic testing can be crucial not only to an EOC patient’s treatment following first-line platinum chemotherapy, but to inform family members of their potential risks as well.

The objective of this project is to understand the observed concordance between ovarian cancer survivors' self-reported genetic testing results and the medical record genetic testing report. The Ovarian Health & Lifestyle Study (OHLS) follows newly diagnosed ovarian cancer patients to understand how lifestyle factors (e.g. exercise, diet, and supplement use) impact quality of life and long-term survival. We collect data using self-report questionnaires administered to participants across various timepoints. Data on genetic testing status, understanding of test results, and mutations in the BRCA1 and BRCA2 genes were collected. Medical record data was manually abstracted using a structured abstraction tool to obtain information on germline genetic testing status and results.

Self-reported genetic testing was compared to the medical record data to calculate the observed concordance. There was 94% agreement between self-report and medical record receipt of genetic testing, including 159 questionnaires from 68 participants. Among the 61 EOC survivors, 58 (95%) reported ever having received genetic testing, with 50 (86%) of those participants reporting ever knowing what genes they were tested for. Moving forward, we plan to conduct additional investigations into the barriers and facilitators that may impact ovarian cancer patients' understanding of their genetic profiles.

Introduction: Therapeutic efficacy in triple-negative breast cancer (TNBC) is limited by reduced immune cell infiltration and drug accumulation—consequences of high heterogeneity and poor vasculature. Focused ultrasound (FUS) is an emerging non-invasive localized therapy that delivers high energy or temperatures at a site causing cell death. FUS modulates tumor vasculature and eventual oxygenation and immune response in tumors. These effects are transient and thus maximize treatment efficacies, the timing of FUS-mediated vascular must be understood. This study uses a novel, non-invasive multi-spectral optoacoustic tomography (MSOT) technique to examine FUS-mediated, spatiotemporal changes in tumor oxygenation and perfusion. Dynamic Contrast Enhanced (DCE)-MSOT quantifies tumor perfusion based on wash-in (NKtrans) and wash-out (kep) pharmacokinetic parameters of an injected FDA-approved indocyanine (ICG) contrast agent (ICG). Oxygen-Enhanced (OE) MSOT quantifies relative oxygen saturation (%SO2) and available oxygen capacity (∆sO2)—a measure of hypoxia. Herein, we use OE-DCE/CE MSOT for a quantitative, non-invasive assessment of tumor vascular perfusion and oxygenation changes in the 4T1 TNBC model in response to different FUS treatments

Methods: Thermal ablation and hyperthermia via magnetic resonance guided (MRg)-FUS were performed in 4T1 tumor bearing mice (Day 0) and OCE/CE MSOT imaging was performed. Optoacoustic (OT) analysis was performed in viewMSOT (iThera Medical). OT images were reconstructed using a filtered back projection algorithm and light propagation model to approximate true optical absorption and correct raw signal amplitude at depth. Signal constituents Hb, HbO2, and ICG were spectrally unmixed to calculate %SO2air, %SO2O2, ∆SO2, and ICG signal. Regions of interest were manually drawn around the tumor area and a well-vascularized region near the spine. Average mean pixel intensity (arbitrary units) values for each biomarker were acquired for tumor regions on day-2, 5 hours post-FUS, day 2, and day 5.

Results: Two MRg-FUS treatment groups were established: hyperthermia (10% of tumor volume exceeded 50ºC) and ablation (30% of tumor volume exceeded 50ºC). Tumor perfusion in both groups was determined by a positive correlation between oxygen saturation change and ICG signal change in response to an oxygen challenge. Hyperthermic tumors demonstrated marked increased perfusion and ∆sO2 within 5 hours post-treatment which eventually decreases by day 5. In contrast ablated tumors displayed decreased ∆sO2 that continued to decline up to 5 days post treatment

Conclusions: OE-DCE/CE MSOT imaging allows accurate assessment of spatial and temporal differences in tumor oxygenation and vasculature resultant of different FUS treatment modalities

Endometrial cancer (EC) is the most common gynecological cancer among women in the United States. Endometrioid EC accounts for over 90% of EC cases, with obesity being a major risk factor due to excess estrogen (E2) production. E2 signals via estrogen receptor alpha (ER) to promote endometrial proliferation and is oncogenic. In contrast, progesterone (P4) signals via progesterone receptor (PR) and counteracts E2-driven growth, functioning as a tumor suppressor. The molecular mechanisms behind PR-mediated suppression of ER signaling are poorly understood. PR has two major isoforms, PR-A and PR-B, that are expressed in normal endometrium and differ by an additional N-terminal 164 amino acids in PR-B. We aim to understand the molecular mechanisms and roles of PR isoforms in suppressing ER activity.

Human ER-expressing EC cell lines, Ishikawa and HCI-EC-23, were engineered to express doxycycline-inducible PR-A, PR-B, both PR-A and PR-B, or control LacZ. RNA-seq and ChIP-seq were performed in hormone-deprived cells treated with vehicle, E2, P4, or combination E2+P4. The single isoform expressing cell lines have been molecularly profiled, while the dual-isoform expressing lines are in development and will allow for comparison to isoform-specific lines.

PR-expressing EC cell lines demonstrated P4-induced growth suppression with a stronger effect in PR-B expressing cells. In Ishikawa cells, P4 treatment led to hundreds of highly similar differentially expressed genes (DEGs) in PR-A or PR-B expressing cells, with PR-B expressing cells having more DEGs. Combination treatment significantly reduced the expression of E2-regulated genes, including the potent oncogene MYC. These results indicate that liganded PR opposes ER’s capacity to regulate transcription, explaining P4’s ability to reduce EC growth. ChIP-seq revealed that active PR redistributes ER across the genome. Nearly half of all ER-bound sites are significantly altered in the combination treatment compared to E2 treatment alone, with a stronger effect in PR-B expressing cells. E2-responsive genes that are suppressed by combination E2+P4 treatment are located near ER-bound regions that are also reduced by combination treatment. This suggests that loss of ER binding near these genes reduces their expression. Together, these data suggest that the transcriptional suppression of ER target genes by PR activity is, at least in part, due to the genomic relocation of ER away from its target genes. We anticipate profiling of the PR-A/PR-B dual-isoform expressing lines to replicate these findings, while also revealing the action of the PR-A/PR-B heterodimer, allowing for a more complete, physiologically relevant understanding of PR isoform action.

Endometrial cancer (EC) is the most common gynecological cancer among women in the United States. Endometrioid EC accounts for over 90% of EC cases, with obesity being a major risk factor due to excess estrogen (E2) production. E2 signals via estrogen receptor alpha (ER) to promote endometrial proliferation and is oncogenic. In contrast, progesterone (P4) signals via progesterone receptor (PR) and counteracts E2-driven growth, functioning as a tumor suppressor. The molecular mechanisms behind PR-mediated suppression of ER signaling are poorly understood. PR has two major isoforms, PR-A and PR-B, that are expressed in normal endometrium and differ by an additional N-terminal 164 amino acids in PR-B. We aim to understand the molecular mechanisms and roles of PR isoforms in suppressing ER activity.

Human ER-expressing EC cell lines, Ishikawa and HCI-EC-23, were engineered to express doxycycline-inducible PR-A, PR-B, both PR-A and PR-B, or control LacZ. RNA-seq and ChIP-seq were performed in hormone-deprived cells treated with vehicle, E2, P4, or combination E2+P4. The single isoform expressing cell lines have been molecularly profiled, while the dual-isoform expressing lines are in development and will allow for comparison to isoform-specific lines.

PR-expressing EC cell lines demonstrated P4-induced growth suppression with a stronger effect in PR-B expressing cells. In Ishikawa cells, P4 treatment led to hundreds of highly similar differentially expressed genes (DEGs) in PR-A or PR-B expressing cells, with PR-B expressing cells having more DEGs. Combination treatment significantly reduced the expression of E2-regulated genes, including the potent oncogene MYC. These results indicate that liganded PR opposes ER’s capacity to regulate transcription, explaining P4’s ability to reduce EC growth. ChIP-seq revealed that active PR redistributes ER across the genome. Nearly half of all ER-bound sites are significantly altered in the combination treatment compared to E2 treatment alone, with a stronger effect in PR-B expressing cells. E2-responsive genes that are suppressed by combination E2+P4 treatment are located near ER-bound regions that are also reduced by combination treatment. This suggests that loss of ER binding near these genes reduces their expression. Together, these data suggest that the transcriptional suppression of ER target genes by PR activity is, at least in part, due to the genomic relocation of ER away from its target genes. We anticipate profiling of the PR-A/PR-B dual-isoform expressing lines to replicate these findings, while also revealing the action of the PR-A/PR-B heterodimer, allowing for a more complete, physiologically relevant understanding of PR isoform action.