Browsing by Subject "Tamoxifen"

Protein-ligand interactions can be detected and quantified using protein folding stability measurements. Thus, protein folding stability changes are closely linked to protein function and are an important biophysical measurement. Stability of Proteins from Rates of Oxidation (SPROX) is one approach for making proteome wide stability measurements on proteins. Previous SPROX data analysis strategies relied heavily on visual inspection of the data to identify protein stability changes. This created a bottleneck in the analysis and chances for human error. As part of this work, several new data analysis strategies were evaluated using data from previously reported ligand-binding studies. One strategy, the so-called difference analysis, was determined to be the data analysis strategy of choice as it minimized false positive and negatives. The difference analysis strategy was applied to identify protein targets of the breast cancer therapeutic tamoxifen (TAM) and its active metabolite 4-hydroxytamoxifen (4OHT) in yeast and protein targets of TAM and its most abundant metabolite n-desmethyl tamoxifen (NDT) in MCF-7 cell lysate. Several yeast protein targets of TAM and 4OHT were identified using SPROX, and they were subsequently validated using a pulse proteolysis strategy. The proteins in an MCF-7 cell line were probed for TAM- and NDT-induced stability changes using the SPROX in combination with two different quantitative proteomics strategies. Together the SPROX experiments on the proteins in an MCF-7 cell lysate enabled over 1000 proteins to be assayed for TAM- and NDT- induced protein stability changes. Ultimately, a total of 163 and 200 proteins with TAM- and NDT-induced stability changes were identified, respectively. A subset of 33 high confidence hits, including those identified using both proteomics strategies or those identified with multiples peptide probes, were assessed for experimental links to the ER using a STRING analysis. One high confidence protein hit, Y-box binding protein 1 (YBX1), was recently shown to bind the estrogen receptor, which is the known target of TAM. Preliminary results generated here using pulse proteolysis and a purified recombinant YBX1 protein construct suggest that YBX1 is a direct protein target of TAM. Proteins with altered expression levels with TAM and NDT treatment were also identified here. In total, 799 and 671 proteins were probed for TAM- and NDT- induced expression changes, respectively, and 49 and 30 proteins had altered expression. Out of the 49 and 30 proteins with TAM- and NDT- induced expression changes, 14 and 4 proteins had TAM- and NDT- induced altered stability, respectively. In addition to the above ligand-binding studies, SPROX was utilized to characterize the stability of the allergen containing proteomes of the European house dust mite, timothy grass pollen, and ragweed pollen. It was determined that the protein allergens in these proteomes were more stable and more abundant (based on transcriptomic data), than non-allergen proteins from these sources.

Luminal breast cancer is among the most prevalent diseases in women in the United States and worldwide. Historically, luminal cancers have been effectively managed with treatments that alter the availability of estrogens or their interaction with the estrogen receptor (ER). Among these, the selective estrogen receptor modulator (SERM) tamoxifen has been widely used. Being a SERM, tamoxifen can function as either an agonist or an antagonist of ER, depending on the cellular context. Within luminal breast cancers, tamoxifen initially behaves as an antagonist. Tamoxifen also has the added benefit of being an exceptionally well tolerated drug, demonstrating a minimal side effect profile.

While current first line therapeutics demonstrate considerable efficacy as treatments for luminal tumors, experimental and clinical evidence indicates that resistance to these therapies is seemingly inevitable for most patients. Mechanisms of resistance involve processes which are both dependent on and independent of the action of ER. Current drug development has focused on improving the ability of pharmacological agents to directly inhibit ER activity. As such, it stands to reason that the identification of therapeutics that may be paired with ER targeted therapies, or which are effective in the setting of resistance, remains critical.

To address the need for new approaches to treat endocrine therapy resistant disease, we have utilized both in vivo and in vitro derived models of aggressive luminal breast cancer to define potential mechanisms leading to diminished and altered drug response. Integrative analysis of the genome, epigenome and transcriptome of one of these models, TamR, has indicated that in the face of substantial selective pressure, luminal breast cancer cells develop epigenetic and genetic alterations that result in the reprogramming of the transcription factor landscape. Specifically, we have observed that luminal breast cancer cells exhibit a unique genome-wide binding profile of lineage determining transcription factor, FoxA1, and its collaborating transcription factor, GRHL2, the function of which defines a transcriptional program associated with decreased response to tamoxifen therapy.

The alteration of various cell intrinsic factors downstream of this reprogramming ultimately influences how these cells respond to and interact with the surrounding microenvironment, the result of which further impacts drug response. We demonstrate that one factor regulated by GRHL2, the Ly6/PLAUR containing protein 3 (LYPD3) is increased in abundance in the setting of endocrine resistance. Additionally, we further demonstrate that the presence of extracellular mitogenic factors, including fibroblast growth factors (FGF) and anterior grade protein 2 (AGR2), alters the transcriptional program of breast cancer cells as well as the activity of cancer associated fibroblasts, respectively.

We further extended our investigation to define aspects of the tumor microenvironment impacting treatment outcomes in luminal breast cancer using mouse models of breast cancer. Previously we observed that the presence of the endogenous SERM, 27-hydroxycholesterol, significantly accelerated the onset and growth of luminal breast cancers. Herein we demonstrate that 27HC derived from macrophages may be responsible, at least in part, for altering the growth of luminal tumors.

The results of these studies provide a comprehensive analysis of alternative pathways that are active in the setting of luminal breast cancer that ultimately alter treatment response. The critical assessment of these pathways has resulted in the indication of potential drug targets in this context, as well as in preclinical testing of alternative treatment approaches. Continued investigation in this vein may provide rational drug combinations for the treatment of advanced luminal breast cancer patients.

Estrogens and the estrogen receptor (ER) have been implicated in the etiology of breast cancer and osteoporosis. However, the mechanisms by which this receptor-ligand complex manifest their regulatory activities in these processes is not completely understood. The development and subsequent definition of the molecular mechanism of action of selective ER modulators (SERMs), compounds with differential relative agonist/antagonist activity, has uncovered an unanticipated complexity in this signaling pathway. Furthermore, these analyses indicat that it is likely that in addition to the classical steroidal estrogens, which exhibit agonist properties, endogenous compounds exist that interact with ER and function as physiological SERMs. Recently, 27-hydroxycholesterol (27HC) was identified as an endogenous ER ligand with tissue-specific estrogenic/anti-estrogenic activities. Indeed, we determined that 27HC exhibited the three basic properties of a SERM: 1) it bound competitively with estradiol (E2) to both genetic subtypes of ER, ERα and ERβ; 2) it induced a unique conformation of ER that is likely related to its biological activity; and 3) it displayed tissue-specific ER modulatory activity in the cardiovascular system, breast, and bone. In particular, we undertook a series of in vivo studies to show that a pathological elevation of 27HC was associated with decreased bone quantity, an effect that was partially rescued by E2 supplementation. The ability of 27HC to decrease bone density in the absence of endogenous estrogens suggests that the circulating level of 27HC may be of critical importance in determining osteoporosis risk in post-menopausal women. Interestingly, cholesterol-lowering statins have been shown to improve bone density; thus, given the stoichiometric relationship between circulating cholesterol and 27HC, our data provide a possible explanation for the observed bone sparing actions of this class of drugs.

In general, it is considered that SERM activity can be explained by the ability to induce differential alterations in ER structure and the impact that this has on the recruitment of functionally distinct cofactors. The results of our studies reveal a much more complex picture and suggest that some SERM pharmacology can be ascribed to actions in pathways that do not include ER. Specifically, we have determined that the SERM 4-hydroxy-tamoxifen (4OHT) can bind to and activate the aryl hydrocarbon receptor (AHR). Given that AHR controls the expression of E2-metabolizing enzymes, our finding that 4OHT regulates AHR in the context of breast cancer could have important pharmacological and pathological implications. Interestingly, our preliminary in vitro data indicate that the ability of 4OHT to inhibit osteoclast (OC) differentiation, and thus aid in preserving bone density in post-menopausal women, is primarily dependent on expression of AHR, not ER. Conversely, the inhibitory activity of raloxifene (RAL), another SERM, on OC differentiation was absolutely dependent on ER. Thus, the activity of 4OHT in bone is likely to be a composite response requiring its actions on both ER and AHR.

Many new aspects of the estrogen and ER signaling pathways have been uncovered as we learn more about ligands that modulate ER by altering its conformation and thus its ability to engage in protein-protein interactions. Collectively, our findings demonstrate that the intersection between cholesterol metabolism, ER signaling, and the AHR pathway will have important consequences in regulating cellular function, and may be involved in the development or progression of multiple disease states.