TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

There is a strong association between estrogen exposure and breast cancer risk. Estrogen can activate estrogen receptor alpha (ERalpha) to increase cell proliferation. Estrogen can also be metabolized into genotoxic compounds to induce DNA damage and mutations. Activation of the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2 like 2 (NFE2L2; NRF2) can alter the production of genotoxic estrogen. The present thesis investigated the signalling mechanisms of ERalpha, AHR, and NRF2 and how their interaction might modulate breast cancer risk. In Chapter 2, genome-wide, but promoter-focused analysis of ERalpha binding sites in T-47D breast cancer cells identified potential cell line specific differences in estrogen signalling between T-47D and the commonly used MCF-7 breast cancer cells. CYP2B6 was identified to be an ERalpha target gene in T-47D cells but not MCF-7 cells, supporting cell line dependent effect in estrogen signalling. In Chapter 3 and 4, genome-wide analyses of AHR binding sites were performed to investigate the molecular criteria governing genomic AHR transactivation in vivo in mouse and in vitro in MCF-7 breast cancer cells. Our analysis identified 1) the previously established aryl hydrocarbon response element to be an important, but not an absolute requirement in AHR transactivation and 2) key epigenetic modifications that modulate AHR-dependent gene regulation. Lastly, in Chapter 5, interaction among ERalpha, AHR, and NRF2 was presented at the regulatory region of two NRF2 target genes, NADPH Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase 1 (HMOX1). ERalpha repressed, whereas AHR enhanced NRF2-dependent NQO1 and HMOX1 mRNA expression through altered p300 recruitment and Histone H3 Lysine 9 acetylation. Collectively, this thesis examined novel molecular mechanisms that might alter breast cancer development/progression by modulating ER, AHR, and NRF2 activity.

There is a strong association between estrogen exposure and breast cancer risk. Estrogen can activate estrogen receptor alpha (ERalpha) to increase cell proliferation. Estrogen can also be metabolized into genotoxic compounds to induce DNA damage and mutations. Activation of the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2 like 2 (NFE2L2; NRF2) can alter the production of genotoxic estrogen. The present thesis investigated the signalling mechanisms of ERalpha, AHR, and NRF2 and how their interaction might modulate breast cancer risk. In Chapter 2, genome-wide, but promoter-focused analysis of ERalpha binding sites in T-47D breast cancer cells identified potential cell line specific differences in estrogen signalling between T-47D and the commonly used MCF-7 breast cancer cells. CYP2B6 was identified to be an ERalpha target gene in T-47D cells but not MCF-7 cells, supporting cell line dependent effect in estrogen signalling. In Chapter 3 and 4, genome-wide analyses of AHR binding sites were performed to investigate the molecular criteria governing genomic AHR transactivation in vivo in mouse and in vitro in MCF-7 breast cancer cells. Our analysis identified 1) the previously established aryl hydrocarbon response element to be an important, but not an absolute requirement in AHR transactivation and 2) key epigenetic modifications that modulate AHR-dependent gene regulation. Lastly, in Chapter 5, interaction among ERalpha, AHR, and NRF2 was presented at the regulatory region of two NRF2 target genes, NADPH Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase 1 (HMOX1). ERalpha repressed, whereas AHR enhanced NRF2-dependent NQO1 and HMOX1 mRNA expression through altered p300 recruitment and Histone H3 Lysine 9 acetylation. Collectively, this thesis examined novel molecular mechanisms that might alter breast cancer development/progression by modulating ER, AHR, and NRF2 activity.

TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

In situ protein detection in human tissues using antibodies reveals the cellular protein localization, and affinity-based proteomic studies can help to discover proteins involved in the development of diseases. However, antibodies often suffer from cross-reactivity, and the lack of positive and negative tissue controls for uncharacterized proteins complicates the mapping of the proteome. The aim of this thesis is thus to improve the methodology for validating antibodies used for immunostaining on formalin-fixed paraffin-embedded tissues. Two of the papers include comparisons between mRNA-expression and immunostaining of corresponding protein. In paper I, ISH and IHC staining patterns were compared on consecutive TMA-slides. The study of well-characterized genes showed that ISH could be used for validation of antibodies. ISH was further used for antibody evaluation, and could validate four out of nine antibodies showing potentially interesting staining patterns. In paper III, transcriptomic data generated by RNA-sequencing were used to identify tissue specific expression in lymphohematopoietic tissues. An increased expression in one or more of these tissues compared to other tissue types was seen for 693 genes, and these were further compared to the staining patterns of corresponding proteins in tissues. Antibody labeling is necessary for many immunoassays. In paper II, two techniques for antibody-biotinylation were compared, aiming to find a stringent labeling method for antibodies used for immunostaining on TMAs. The ZBPA-method, binding specifically to Fc-part of antibodies, was found to be superior to the Lightning Link-biotinylation kit targeting amine groups, since labeling of amine groups on stabilizing proteins in the antibody buffer causes unspecific staining. The localization of the estrogen receptor beta (ERβ) in human normal and cancer tissues was studied in paper IV. Thorough evaluation of 13 antibodies using positive and negative control cell lines showed that only one antibody, PPZ0506, is specific for ERβ in all three immunoassays used. Contradictory to previously published data, tissue profiling using PPZ0506 showed that ERβ is expressed in a limited number of normal and cancer tissues. In conclusion, the present investigations present tools for validation of antibodies used for large-scale studies of protein expression in tissues.

Breast cancer is a heterogeneous disease that afflicts all patients differently, and therefore requires individualized treatment depending on a large variety of factors. Several methods of classification exist to divide patients into meaningful groups in order to better personalize their treatment regimens. Healthcare is evolving into more use of personalized treatments that can more effectively treat patients on an individual level, rather than by using more generalized treatments that may not prove effective in all patients. In addition, personalized treatment also aims to reduce adverse effects, while increasing effectiveness. Estrogen receptor (ER) status is one such method of grouping breast cancer patients into different treatment groups. Based on stage diagnosis and determination of receptor status, initial treatments such as surgery or radiotherapy may be used. Standard chemotherapy is another method, however, side effects may vary among patients and may be quite adverse. Other treatments include hormone or receptor blocking. This thesis has identified an alternatively spliced variant of classical ERα that resides in the plasma membrane of breast cancer cells and plays a major role in rapid signaling by estrogen. The overall aim of this thesis was to examine the role of the membrane receptor for 17β-estradiol (E2) in breast cancer that enhances breast tumor aggressiveness and to evaluate the mechanisms by which it functions. The general hypothesis was that nonclassical estrogen signaling through the proposed membrane-associated ER, ERα36, can promote breast tumor aggressiveness by enhancing cell survivability while altering expression of angiogenic and metastatic factors. This work examined the mechanisms of ERα36-dependent signaling in breast cancer cells, and the correlation of ERα36 to clinical outcome in human breast cancer tissue through histological evaluation. These data provide significant research as they provide a greater understanding of estrogen signaling in breast cancer through ERα36 and its role in tumorigenicity and metastasis. This study also proposes further clinical examination of ERα36, and suggests drug design to target ERα36 followed by preclinical studies to determine if drugs targeting ERα36 would benefit breast cancer patients by reducing tumorigenicity and increasing survival.

Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2. Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland. Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.

Beyond their physiological roles, nuclear receptors have been exploited for their ability to act as intracellular sensors of small molecules. Accordingly, yeast two- and three-hybrid systems have been developed, exploiting them to control reporter gene expression. These systems may be used to identify nuclear receptor ligand interaction, or for protein engineering applications, particularly of the nuclear receptor ligand binding domain. In this work, the use of estrogen receptors as sensors for enzyme catalysis is explored, where expression of a reporter gene is induced in the presence of the product from an enzymatic reaction. This system, which we have called enzyme-activated growth, has applications for the engineering of biocatalysts. Biocatalytic routes are currently being explored in industrial applications since they often have financial and environmental benefits over traditional heterogeneous catalysis. Enzyme-activated growth is designed to serve as a system to select for engineered enzymes capable of catalyzing the desired reaction. For this work, a new yeast two-hybrid strain has been developed and characterized to allow for detection of both agonist and antagonist compounds. To increase the sensitivity of this assay, a variant of the estrogen receptor was created through random mutation, which responded to ligand concentrations an order of magnitude lower than the wild type receptor. The five mutations identified in the best variant were previously unknown in the literature and the roles of each of these are investigated, as is the mechanism by which they alter ligand sensitivity. As a proof-of-principle, the enzymatic production of genistein, an estrogenic metabolite from plants, using the enzyme isoflavone synthase, as well as the production of estrogen from testosterone, is explored. Synthesis of genistein from the starting material naringenin in vivo was detected in the yeast two-hybrid strain; however, attempts at pairing this with estrogen receptor activation and cell growth were met with limited success. Lastly, targeting the estrogen receptor with a series of novel anti-cancer therapeutics is explored. These compounds were designed to both bind and (in)activate the estrogen receptor while inhibiting histone deacetylase activity. The (anti-)estrogenic properties were analyzed as well as their potency as histone deacetylase inhibitors. These properties were compared to their anti-proliferative effects against various cancerous and healthy cell lines to determine their potential as selective anti-cancer therapeutics.

Hormone replacement therapy and estrogen replacement therapy have shown the ability to reduce risk of colon cancer development in clinical and animal studies, but in vitro research has been unable to reproduce an estradiol (E2) induced response in colon cancer cell lines. We demonstrated that young adult mouse colonocytes (YAMC, non-malignant colonocytes) exhibit an anti-proliferative response to E2 treatment. These cells demonstrate reduced cell culture growth and increased apoptosis in response to E2. YAMC cells containing an activated Ras mutation are considered to be malignantly transformed, and lose the ability to respond to E2 treatment. Fulvestrant (ICI) was used as an estrogen receptor antagonist to determine that these results were estrogen receptor mediated. Furthermore, this effect was demonstrated to require the presence of ER? through the use of a transgenic ERbeta knockout mouse. In these mice, the presence of E2 significantly reduced the formation of azoxymethane induced premalignant lesions. Since YAMC cells exhibit an anti-proliferative response to E2 treatment, we utilized isogenic YAMC cell lines with and without a dominant negative p53 mutation to demonstrate that this E2 induced action involves p53 activity. E2 treatment results in increased p53 transcriptional activity and a pro-apoptotic change in expression of p53 downstream targets. Presence of the dominant negative p53 mutant nullifies these effects of E2 treatment. The involvement of p53 in the previously described protection against AOM induced premalignant lesions, was investigated using wild type and heterozygous p53 knockout (Het p53KO) mice. The reduction in p53 protein corresponded to reduced effectiveness of E2 treatment on the prevention of premalignant lesion formation in Het p53KO mice. In summary, our data indicate that E2 treatment induces anti-proliferative responses in non-malignant colonocytes and protects against the formation of carcinogen-induced premalignant lesions. These effects require the presence of functional ER? and p53. Further studies are required to more thoroughly elucidate the specific interactions and downstream effects of ER? and p53 in response to E2 stimulation.