Cancer models have been utilized to define properties of the anti-tumor response and factors that regulate the function of distinct cellular populations. The overarching results highlight the importance of the Wnt signaling pathways that utilize the canonical pathways but also new pathways that don‚Äôt utilize the canonical pathways. These pathways may also be significantly impacted by Notch signaling. Novel cellular functions have been identified and the hope is that it will lead to innovative therapeutic approaches to treat cancer. In addition, studies of the Wnt antagonist DKK1 in the chronic parasitic infections by Leishmania major are being utilized to define the primary mechanistic interactions that lead to the infections.
The current research interests of Professor Bothwell in the Immunobiology Dept. began with studies of intestinal tumorigenesis using the APC/Min+ mouse model and have given insight into regulation of anti-tumor immune responses. Homozygous deletion of the cytokine IL-17A in APC/Min+ mice had a profound effect on small intestinal polyps. Further characterization of the Wnt ligands in APC/Min+ mice in many cell types demonstrated regulatory T cells(Treg) have high expression of the Wnt antagonist Dickkopf1 (DKK1) on the cell surface that is required for Treg function and is also a very potent driver of type 2 immune responses. Ablation of type 2 responses by deletion of Stat6 lead to potent effects on myeloid derived suppressor cells (MDSC) and activation of a CD8 T cell response to the tumor. The effect on MDSC can be dramatically altered by an inhibitor of RIPK3 which results in induction of a Th17 T cell response. DKK1 is expressed at very high levels in many solid tumors averaging about a 1000-fold increase over normal tissues in public data bases of over 70,000 solid tumors. Thus it is a potent driver of cancer in solid tumor types. Studies on another DKK family member, DKK2, by conditional deletion in intestinal epithelial or intestinal stem cells showed a significant reduction in colon tumors in a standard AOM-DSS (Azoxy methane-dodecyl sulfate sodium) tumor model. RNAseq studies of polyps using Ingenuity Pathway Analysis (IPA) suggested that the transcription factor HNF4ÔÅ° was critical to tumor formation. This lead to identification of a signaling pathway using intestinal organoids in which DKK2 protein can stimulate transcription of DKK2 which ultimately results in activation of c-Src by Y416 phosphorylation. This then leads to phosphorylation of 3 Y residues on HNF4ÔÅ°1 which then targets its degradation by the proteasome and results in reduction of the stem cell marker Lgr5. This then becomes at least one mechanism by which c-Src can promote to colorectal cancer. A splenic injection model was then utilized to test metastasis of mutant APC, Kras, P53 (AKP) organoids to liver via the portal vein. Indeed these mutant AKP organoids readily metastasized to liver while the DKK2 knockout AKP organoids were completely inhibited from metastasis. The organoids expressed a tdTomato fluorescent protein marker which revealed that a significant percentage of the metastasized lymphoid cells contained tumor antigen from the original organoids plus several hemopoietic antigens. This has lead to the hypothesis that once a cancer stem cell can mutate it may interact with hemopoietic cells which can result in trogocytosis and generation of some hybrid cells. This was observed with APC mutant only organoids and not WT organoids, In vivo there is a strong selection for survival even though most such cells will die. Such a hybrid cell results in considerable transcriptional reprogramming and induction of genetic instability (e.g., aneuploidy, chromothripsis or tandem duplication phenotype). A small number of such cells may lead to aggressive tumor formation and metastasis. Further characterization of DKK2 deficiency will address the effects on formation of a suitable tumor microenvironment for development of metastatic sites. This suggest that cancer stem cells and hemopoietic cells in a patient can lead to fusion and tumor formation. Patient derived xenograft models (PDX) in this way can be utilized to access mechanisms of tumor formation and metastasis and optimize effects of therapeutic treatments for a given patient. They can also identify critical differentiation pathways for basic development of cancer. Attempts are being made to expand patient neoantigen specific T cells to tumors for direct therapeutic treatment to kill a tumor. This will be extended to multiple patients and with several different tumor types. Separate experiments will address the nature of the molecular interactions involved in the induction of a chronic infection with Leishmania major chronic infection in BALB/c mice.