Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 91-124).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Campus access only. Vita. Bibliography: 176-188.

Self-renewing, chemoresistant cancer cells that contribute to cancer metastasis and patient relapse have properties similar to those of stem cells, and have been termed "cancer stem cells" (CSCs) in the literature. The identification of signaling pathways that regulate CSC development and/or function is an important step towards understanding why patients relapse, and towards development of novel therapeutics that specifically target CSC vulnerabilities. Recent studies have identified a role for the aryl hydrocarbon receptor (AHR), an environmental carcinogen receptor implicated in cancer initiation, in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in CSC development. To test this hypothesis, AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells was modulated with AHR ligands, shRNA, or AHR-specific inhibitors and their phenotypic, genomic, and functional CSC characteristics were evaluated. Aldehyde dehydrogenase (ALDH) was used as an epithelial stem cell marker for flow cytometry. Results demonstrate that: 1) ALDHhigh cells express elevated levels of Ahr and the AHR-driven gene that encodes cytochrome p450 isoform 1b1 (Cyp1b1), 2) AHR knockdown reduces ALDH activity, 3) AHR hyper-activation significantly increases ALDH1 activity, expression of stem cell- and invasion/migration-associated genes, and accelerates cell migration, 4) a highly significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of the CSC- and invasion/migration-associated gene sets was seen with genomic data obtained from 79 human breast cancer cell lines and over 1850 primary human breast cancers, 5) the AHR interacts directly with the transcription factors Sox2 and Runx1, and AHR ligands increase this interaction, 6) AHR knockdown inhibits tumorsphere formation in low adherence conditions, 7) AHR inhibition blocks the rapid migration of ALDHhigh cells and reduces ALDHhigh cell chemoresistance, and 8) AHR knockdown inhibits tumor growth and reduces tumor Aldh1a1, Sox2, and Cyp1b1 expression in orthotopic xenografts. These data suggest that the AHR plays an important role in development of CSCs in a large fraction of human breast cancers and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of CSC-like properties.

Cancer and thrombosis are common co-occurrences in healthcare today. Cancer is the second leading cause of death in the United States with thrombosis being the second leading killer of cancer patients behind tumor progression. Cancer patients as a whole are 4 to 7 times more likely to develop thrombosis and 20%-30% of all first-time thrombosis diagnoses are cancer-related. Risk assessment and treatment options have much room for improvement. The lack of success for conventional antithrombotic prophylaxis suggests that hyperthrombosis in cancer works through a discrete pathway. A discovery by our group in recent years correlated the increased activity of Aryl hydrocarbon receptor (AHR) in the body to increase thrombotic phenotypes in patients with chronic kidney disease. Another group had published a manuscript about Kynurenine (Kyn), an activating ligand of the receptor that was produced by tumor cells to promote tumor growth through an AHR pathway (Opitz 2011). The link between the findings of these two groups could show that Kyn—AHR pathway is causing the increase in thrombosis in cancer patients. We used an animal model of thrombosis in cancer and created a new variation of it to test the Kyn—AHR pathway. We hypothesized that cancerous animals would show an increase in thrombosis and increased levels of AHR and Kyn along with downstream elements such as Tissue Factor (TF). Cancer was induced on nude mice via xenograft injection of cancer cells and 4-5 weeks of incubation to allow the tumors to proliferate. After the incubation period, mice underwent inferior vena cava (IVC) ligation, and were then euthanized 48 hours later. Two types of cancer were tested: HT-29 colon adenocarcinoma and A549 non small cell lung adenocarcinoma. There were 4 animal groups: mice that were injected with cancer cells and operated on, mice that were injected with cancer cells but not operated on, mice that were not injected but were operated on, and mice that did not receive neither the injections or the operation. After euthanasia, blood, tumors, and major organs were harvested to assess markers and pathways of thrombosis associated with cancer. We were able to successfully grow xenograft tumors in the nude mice. The HT-29 tumors grew very aggressively while A549 tumors experienced a small latent period before starting to proliferate. Animals with HT-29 and A549 xenograft tumors displayed greater thrombosis, measured by the weight of the blood clot formed in the IVC due to ligation (p=0.04 and p=0.05, respectively). HT-29 also displayed significant increases in Kyn, AHR activity, indoxyl sulfate (IS), and showed increased staining of tissue factor with immunohistochemistry. A549 did not have significant p-values in these experiments, but did show upward trends in all categories besides IS sera levels. In summary, we developed a new animal model of thrombosis in colon adenocarcinoma and showed significant increases in thrombosis as well as multiple markers of thrombosis. This is an exciting and complex way to study thrombosis in cancer in an in vivo approach with opportunities for future therapeutic testing. / 2020-07-03T00:00:00Z

The main goal of this thesis was to develop new methods and knowledge that will explain and predict species differences in sensitivity to dioxin-like compounds (DLCs) in birds. The important achievements and results obtained from the four experimental chapters of this thesis are summarized as follow: (1) an efficient luciferase reporter gene (LRG) assay was developed for use with 96-well cell culture plates; (2) the results obtained from LRG assay were shown to be highly correlated to available in ovo toxicity data; (3) amino acids at positions 324 and 380 within the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) were shown to be responsible for reduced Japanese quail (Coturnix japonica) AHR1 activity to induce a dioxin-responsive reporter gene in comparison to chicken (Gallus gallus domesticus), and ring-necked pheasant (Phasianus colchicus) AHR1 in response to different DLCs; (4) AHR1 LBD sequences of 86 avian species were studied and differences at amino acid sites 256, 257, 297, 324, 337 and 380 were identified. It was discovered that only positions 324 and 380 play a role in AHR1 activity to induce a dioxin-responsive gene; (5) in COS-7 cells expressing chicken AHR1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) are equipotent inducers of the reporter gene and bind with similar affinity to chicken AHR1, however, in the cells expressing pheasant, Japanese quail and common tern (Sterna hirundo) AHR1, PeCDF is a stronger inducer than TCDD. PeCDF also binds with higher affinity to pheasant and quail AHR1 than TCDD. The results of this thesis show that embryo lethal effect of DLCs in avian species can be predicted by use of two new non-lethal methods: (1) the LRG assay and (2) determination of the identity of the amino acids at positions 324 and 380. The findings and methods described in this thesis will be of use for environmental risk assessments of DLCs.

Pituitary hormones are known to alter sex steroid receptor levels in the liver, and possibly the actions of the steroids as well. Recently, two classes of estrogen binding proteins have been characterized in male rat hepatic cytosol: a high affinity, low capacity estrogen receptor, and a lower affinity, higher capacity sex steroid binding component (moderate affinity component). It is of interest that the moderate affinity component binds both androgens and estrogens. A high affinity, low capacity androgen receptor has not been convincingly demonstrated in rat hepatic cytosol. Therefore, we have investigated the relationship of the moderate affinity component to sex steroid modulation of hepatic aryl hydrocarbon hydroxylase (AHH) activity as a possible control mechanism. Because of the sexual dimorphism for hepatic drug and steroid metabolism known to occur in rat liver, we chose this model to study. We have shown that no sex difference exists for the binding of pH]-estradiol to the estrogen receptor from either immature or adult rats. However, the moderate affinity component does exhibit a sex difference. We did not detect binding to the moderate affinity component in adult female or immature rats of either sex. This site could normally only be measured in the adult male. These findings were consistent with the age and sex dependent elevation of male AHH activity. We have also observed that gonadectomy of the male reduced the levels of AHH activity and the capacity of the moderate affinity component in a testosterone reversible fashion. These results were obtained using either unlabeled estradiol or dihydrotestosterone (DHT) as competitors for [³H]-estradiol binding. Administration of mestranol reduced AHH activity and the capacity of the moderate affinity component in the male. The moderate affinity component was not detected in the pseudoherma-phroditic rat which resembled the female, rather than the male, with respect to control and induced AHH activity. Hypophysectomy of the female resulted in an increase in AHH activity and detection of the moderate affinity component. Hypophysectomy of the male reduced both the capacity of the moderate affinity component and AHH activity. Unlike the gonadectomized male, testosterone had no restorative effect on the levels of AHH activity or the capacity of the moderate affinity component in the hypophy-sectomized rat. Continuous infusion of rat growth hormone (rGH) reversed the effect of hypophysectomy on the increased AHH activity and capacity of the moderate affinity component in the female. Administration of rGH to the hypophysectomized male abolished the detection of the moderate affinity component and reduced AHH activity to control female levels. This suggested rGH may be the pituitary hormone involved in production of the female level of metabolism. The effects of prolactin were not as clear. Therefore, we have demonstrated the modulation of AHH activity by peripheral sex steroids, and the regulation of these parameters by rGH. We have shown, the capacity of the moderate affinity component to vary in a manner that paralleled changes in hepatic AHH activity in different physiological models. Changes in the estrogen receptor were not found to be consistent with changes in AHH activity in these models. We conclude that the moderate affinity component is comparable to the male hepatic cytosolic DHT-binding protein. Furthermore, this component is associated with sex steroid action on hepatic AHH activity in the male rat. Interestingly, we have also shown this component as well as the estrogen receptor, to bind polycyclic aromatic hydrocarbons. Both 3-methylcholanthrene and benzo[a]pyrene competed for [³H]-estradiol binding to the estrogen receptor and moderate affinity component. In addition, dioxin congeners demonstrated specificity for the estrogen receptor in the female. However, this was not observed for the estrogen receptor or moderate affinity component in the male. The significance of this is presently unclear. / Pharmaceutical Sciences, Faculty of / Graduate

No description available.

The aryl hydrocarbon receptor (AhR) carboxyl terminal transcriptional activation domain was cloned, purified in denatured conditions from bacteria, refolded via limited dialysis, and analyzed for proper refolding via co-immunoprecipitation with the known binding partner SRC-1. This AhR NΔ515 transactivation domain construct was used, along with amino terminal AhR deletion constructs AhR CΔ274 and AhR CΔ553, to attempt to elucidate the nature of the interaction between AhR and p23 in vitro.

At the intersection between autoimmune disease and cancer lies a disruption in the balance of our body’s critically important immune system, and, specifically, in its regulation. While autoimmune diseases are the result of overactivation and a failure to regulate improper responses to the body’s own tissues, cancer is the result of improper suppression and a failure to recognize and eradicate transformed malignant cells. Although they are fundamentally different conditions, overlap can be found in the pathways which are critical to disease progression and which may represent important therapeutic targets. One such pathway implicated in both autoimmunity and cancer is the aryl hydrocarbon receptor (AhR). AhR activation suppresses immune cell activation through the modulation of T cell differentiation and antigen presenting cell (APC) function. AhR activation shows a beneficial therapeutic effect in models of autoimmune disease, but has also been implicated in driving cancer progression and tumor-mediated immunosuppression. While it is clear that the AhR plays an important role in the immune response, the mechanisms behind AhR regulation of the immune system and the effects of its modulation in autoimmunity and cancer are still not fully understood. Thus, in this work, we investigated the effect of targeting the AhR in models of autoimmunity and cancer, using the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) and the murine oral cancer (MOC) model of oral squamous cell carcinoma (OSCC). We demonstrated that AhR activation using the endogenous ligand 2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induced a tolerogenic transcriptional response in mouse and human dendritic cells (DCs) associated with the induction of immunoregulatory/immunosuppressive mechanisms. We further showed that targeting the AhR using a nanoliposome (NLP) delivery platform, which co-encapsulated a MS autoantigen, suppressed the development of EAE in multiple models, both in preventative and therapeutic contexts. This disease suppression was associated with the expansion of antigen-specific FoxP3+ regulatory T cells (Treg cells) and IL10+ type 1 regulatory T cells (Tr1 cells), and a reduction in CNS-infiltrating effector T cells (Teff cells). Using the MOC1 model of OSCC we demonstrated that deletion of the AhR in MOC1 malignant cells completely blocks in vivo tumor growth in an immune system-dependent manner and renders mice completely immune to either local or systemic re-challenge with wildtype MOC1 cells. Suppression of tumor growth was associated with a decrease in the expression of suppressive immune checkpoint markers including PD-L1 and CD39 on macrophages, dendritic cells, and Ly6G+ myeloid cells, and PD-1, CTLA4, Lag3, and CD39 on CD4+ T cells. Further, the AhR was found to control expression of chemokines and immunosuppressive IDO and PD-L1 in malignant cells themselves, suggesting that AhR activity in tumor cells may simultaneously regulate multiple immune checkpoints. Taken together, these results provide new insight into the critical role for the AhR in both autoimmunity and cancer, and confirm it as a valid therapeutic target for both diseases. / 2022-03-05T00:00:00Z

No description available.