Tumor Suppressor APC and Musashi1: Double-Negative Feedback, Wnt Signaling and Colon Cancer

Colorectal cancer is the second leading cause of cancer related deaths in the United States. Approximately 80% of all colon cancers are associated with a mutation in the Adenomatous polyposis coli (APC) tumor suppressor gene. We study the intracellular functions of normal APC and how loss of APC function leads to the formation of precancerous polyps in the intestines of mice and humans. The studies presented here are particularly concerned with the role of APC in maintenance of homeostasis in the intestinal epithelium. I have identified a double-negative feedback loop between APC and a sequence specific RNA binding protein, Mushashi-1 (MSI1). I hypothesize that this feedback loop serves to maintain a critical balance and that disruption of this balance leads to loss of homeostasis and ultimately tumorigenesis in the intestinal epithelium. Studies described here and by others have shown that MSI1 is a target of β-catenin transcriptional activation through the canonical Wnt signaling pathway. My work has demonstrated that APC mRNA is a target of MSI1 binding and translational repression. Further study has indicated that MSI1 binding also results in stabilization of the APC mRNA, presumably as a rapid response mechanism to repress Wnt signal transduction once the signal is no longer present. Finally, my studies indicate that MSI1 expression is stimulated by the transcription factor c-MYC, the most commonly deregulated human oncoprotein. Though overexpression of c-MYC stimulates MSI1 expression, c-MYC expression is not stimulated by loss of APC in our cell culture system. The implication is that c-MYC stimulation of MSI1 expression may only occur in tumors with deregulated c-MYC expression. The studies presented here have uncovered novel molecular mechanisms involved in regulation of the canonical Wnt signaling pathway in the intestinal epithelium, thereby expanding the potential methods by which deregulation of this pathway contributes to colorectal carcinogenesis

The role of MYC-EGR1 Pathway in Colorectal Cancer Development and its Relationship to Wnt Signaling

Lexi Chrysochoos, Emily Abdoney, and Erick Spears The Role of MYC-EGR1 Pathway in Colorectal Cancer Development and its Relationship to Wnt Signaling Colorectal tumors show high levels of Wnt pathway activity, most often due to an initiating mutation in critical Wnt pathway components. Wnt pathway activation leads to b-catenin-driven expression of genes that generally stimulate cell cycle progression. One such Wnt pathway target gene is MYC, a well described oncogene involved in many cancers, including colorectal. MYC is known to stimulate proliferation but in certain cellular contexts, can stimulate apoptosis by stimulating the expression of EGR1. This is termed a noncanonical MYC target gene because EGR1 lacks typical MYC binding elements in its promoter. This unique MYC-EGR1 pathway is activated in specific cellular contexts, lacking appropriate p53 activity, often associated with colorectal tumorigenesis. In these experiments, sister HCT116 human colorectal cancer cell lines were treated with shRNA to employ RNA interference targeting MYC or EGR1 to assess the roles these genes might play in colorectal cancer cell proliferation. b-catenin wildtype and mutant HCT116 cells were treated with a non-targeting (scramble, control) shRNA, MYC shRNA, and EGR1 shRNA to specifically and temporarily target the expression of these gene. After transduction, cultures were grown, and cells were counted to assess proliferation of these cell lines with the specific interruptions in gene expression. The goal of our experiment was to evaluate the proliferation of these cells after transduction with targeted shRNAs and assess any impact that MYC and EGR1 have on proliferation in this cellular context. As the MYC-EGR1 pathway has also been shown to stimulate apoptosis in cells lacking functional p53, we also performed an apoptosis analysis and evaluated the propensity of the cell lines to apoptosis. These experiments give further insight into the role of the MYC-EGR1 pathway in colorectal cancer development and its relationship in to the Wnt signaling pathway in colorectal cancer cells

Assessing the Roles of MYC and EGR1 in Endoderm Differentiation

The Wnt signaling pathway is evolutionarily conserved from fruit flies to humans. Strongly associated with development and embryonic morphogenesis, it is known to be required for early endoderm and later hindgut development in mammals. In adults, the Wnt signaling pathway is required for the proper maintenance of the intestinal epithelium and mutations in critical Wnt signaling pathway components are initiating events in the development of essentially all colorectal cancers. An important target gene whose expression is upregulated by Wnt signaling pathway activation is the oncogene MYC. This gene produces a transcription factor whose typical cellular role is to stimulate cell cycle progression and proliferation. Under specific cellular conditions, namely in the absence of functional p53, MYC has been shown to upregulate the expression of EGR1, ultimately leading to apoptosis. Interestingly, we have observed specific expression of these MYC-EGR1 pathway components during the differentiation of human induced pluripotent stem cells (hiPSCs) into definitive endoderm. These studies seek to assess the role of the MYC-EGR1 pathway in early endodermal development. Using immunofluorescence staining, we seek to assess whether MYC and EGR1 are absent from undifferentiated pluripotent stem cells by co-staining for the pluripotency marker OCT-4 and MYC or EGR1. Next, we seek to determine whether MYC, EGR1, or both are required for endoderm differentiation by specifically targeting their expression using shRNA during endoderm differentiation. These studies will contribute to our understanding of the molecular mechanisms involved in endoderm differentiation and the role of the MYC-EGR1 pathway in the process

CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction

MYC and EGR1 Expression and Proliferation of Colorectal Cancer Cell Lines

The American Cancer Society estimates that in 2023, one person every five minutes will be diagnosed with colorectal cancer. An abundance of research has been conducted on the relationship between the Wnt/β-Catenin Signaling Pathway and colorectal cancer. In this pathway, the growth-stimulating protein Wnt binds to a receptor called Frizzled, which transduces a signal. This ultimately results in β-Catenin-driven expression of genes associated with cell proliferation. One such gene is MYC, which is said to be the most important proliferative target gene of the Wnt pathway in colon cancer. While MYC expression is typically associated with cell cycle progression, under certain cellular contexts it has been shown to stimulate apoptosis by stimulating the expression of the tumor suppressor EGR1. Previous literature has suggested that EGR1 is a direct transcriptional target of MYC but does not contain the canonical MYC binding element in its promoter, and its expression is only activated in the absence of other cell cycle control elements. In this project, we seek to determine whether the MYC-EGR1 pathway is active in HCT116 colorectal cancer cell lines with wildtype and mutant CTNNB1 (the β-catenin gene). Using RNA interference, we decreased the expression of MYC or EGR1 and observed proliferation in these cell lines. We also evaluated the expression of MYC and EGR1 in these cells during the proliferation assays to determine if changes in β-catenin activation are impacting these downstream effectors. With these studies, we hope to understand whether the cellular context associated with colorectal cancer activates this unique MYC-EGR1 pathway and whether it changes proliferation in these cells

MYC-EGR1 Pathway in Growth of HCT116 cells With and Without Functional p53

Colorectal cancer is the second most common cause of cancer death across the population. Most colorectal cancers are initiated by mutations in the Wnt-signaling pathway. MYC is a target gene of the Wnt-signaling pathway and a well-described oncogene that is often upregulated in colorectal and other malignancies. Under certain cellular contexts, specifically in the absence of functional p53, MYC is known to stimulate the expression of EGR1 resulting in apoptosis. While the MYC-EGR1 pathway has been studied in rodent cells in a synthetic knockout system, we were interested in understanding the effect of this pathway on human colorectal cancer cells. To assess the role of the MYC-EGR1pathway on the growth of human colorectal cancer cells, we employed HCT116 sister cell lines with and without the functional TP53 gene. The expression of either MYC or EGR1 was specifically and temporarily knocked down using targeting siRNAs for each of the pathway components. Serum starved HCT116 p53+/+ and p53-/- cells were transduced with siRNAs targeting MYC or EGR1 expression and proliferation was assessed. These studies are furthering our understanding of an understudied MYC-EGR1 pathway and its role in colorectal cancer cell cycle

MB0 and MBI Are Independent and Distinct Transactivation Domains in MYC that Are Essential for Transformation

MYC is a transcription factor that is essential for cellular proliferation and development. Deregulation or overexpression of MYC occurs in a variety of human cancers. Ectopic expression of MYC causes hyperproliferation and transformation of cells in culture and tumorigenesis in several transgenic mouse models. Deregulation of MYC can also induce apoptosis through activation of p53 and/or ARF tumor suppressors as a safeguard to prevent tumorigenesis. MYC binds to thousands of genomic sites and regulates hundreds of target genes in a context-dependent fashion to mediate these diverse biological roles. The N-terminal region of MYC contains several conserved domains or MYC Boxes (MB), which influence the different MYC transcriptional and biological activities to varying degrees. However, the specific domains that mediate the ability of MYC to activate transcription remain ill defined. In this report, we have identified a new conserved transactivation domain (TAD), MB0, which is essential for MYC transactivation and target gene induction. We demonstrate that MB0 and MBI represent two distinct and independent TADs within the N-terminal 62 amino acids of MYC. In addition, both MB0 and MBI are essential for MYC transformation of primary fibroblasts in cooperation with activated RAS, while MB0 is necessary for efficient MYC-induced p53-independent apoptosis