Browsing by Author "Bawa-Khalfe, Tasneem"
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Item A Historical Review of Hematopoietic Stem Cell Transplantation: Developmental Advancements Then and Now(2023-04-23) Wade, JoshuaA review of the history of regenerative medicine focusing on the use of hematopoietic stem cells (HSCs) from their initial inception in bone marrow transplantation for blood disorders and diseases to their expanded uses today through advancements in technology. Commonly treated malignant and non-malignant bone marrow transplant conditions include but are not limited to leukemia, non-Hodgkin lymphoma, and aplastic anemia. Bone marrow transplants are performed by autologous or allogeneic transplantation with multipotent HSCs. The first unrelated allogeneic transplant was performed by Dr. Edward Donnall Thomas in 1957. The initial approaches using stem cells as a form of treatment resulted in great difficulty and failure with few exceptions. However, discoveries such as the major histocompatibility complex within humans known as the human leukocyte antigen (HLA), allowed researchers to identify genetic matches between donors and recipients resulting in an increased rate of successful procedures. Since then, advancements in the field have introduced new techniques that improve the efficacy of hematopoietic stem cell transplantation and expanded use to more populations. Such advancements include improved conditioning regimens, matched unrelated donors, umbilical cord HSCs, and more potent immunosuppressants and antimicrobial drugs. For patients that experience tissue damage due to conditioning regimens or cancer malignancy, HSCs use a “homing” mechanism to mobilize to the affected area and release chemical factors that promote tissue recovery. Patients that undergo transplantation are put at risk of several conditions including graft-versus-host disease and opportunistic infections, however, complication-prevention regimens have been put in place to decrease the mortality rate. This literature review serves to roughly gauge how this field of medicine has developed since the 1950s and where future implications lie. Said implications include emerging ways to treat graft-versus-host disease, improved HLA typing matches, and HSC uses in transplantable organs like the liver.Item Androgen Receptor in Hormone Receptor-Positive Breast Cancer(2023-12-29) Khan, Ashfia Fatima; Karami, Samaneh; Peidl, Anthony S.; Waiters, Kacie D.; Babajide, Mariam Funmi; Bawa-Khalfe, TasneemBreast cancer subtypes expressing hormone receptors (HR+ BCa) have a good prognosis and respond to first-line endocrine therapy (ET). However, the majority of HR+ BCa patients exhibit intrinsic or acquired ET resistance (ET-R) and rapid onset of incurable metastatic BCa. With the failure of conventional ET, limited targeted therapy exists for ET-R HR+ BCa patients. The androgen receptor (AR) in HR-negative BCa subtypes is emerging as an attractive alternative target for therapy. The AR drives Luminal AR (LAR) triple-negative breast cancer progression, and LAR patients consistently exhibit positive clinical benefits with AR antagonists in clinical trials. In contrast, the function of the AR in HR+ BCa is more conflicting. AR in HR+ BCa correlates with a favorable prognosis, and yet, the AR supports the development of ET-R BCa. While AR antagonists were ineffective, ongoing clinical trials with a selective AR modulator have shown promise for HR+ BCa patients. To understand the incongruent actions of ARs in HR+ BCa, the current review discusses how the structure and post-translational modification impact AR function. Additionally, completed and ongoing clinical trials with FDA-approved AR-targeting agents for BCa are presented. Finally, we identify promising investigational small molecules and chimera drugs for future HR+ BCa therapy.Item Behavioral, Cognitive, and Biochemical Consequences of Early Life Stress in Later Life: Insights from an Animal Model(2017-12) Liu, Hesong; Salim, Samina; Eikenburg, Douglas C.; Hurd, Yasmin; Eriksen, Jason; Bawa-Khalfe, TasneemAdverse experiences during early life contribute to the development of psychiatric conditions in later life. In fact, young children who directly experience or witness traumatic event(s) during early life, a sensitive developmental period, are considered highly vulnerable to psychiatric disorders during adult life. Interestingly, not all children who experience traumatic events are equally at risk of developing later life psychiatric disorders. Some are resilient despite being exposed to the same risk factors, while others are susceptible. The relationship between early life trauma exposure and development of later life psychiatric symptoms is not fully understood, and the mechanistic basis for resilience is also not clear. Clinical and preclinical studies have suggested that defects in stress-adaptive mechanisms potentially contribute to etiology of later life psychiatric conditions. Preclinical data from our laboratory has indicated poor oxidative/antioxidative balance as a critical component of maladaptive stress responsiveness in rodents. Our published work has demonstrated that induction of psychological stress leads to behavioral and cognitive deficits in rats. These impairments correlate with an increase in oxidative stress markers in the periphery as well as in selected regions of the brain including the hippocampus, amygdala, and the prefrontal cortex. Moreover, heightened oxidative stress was associated with decreased levels of key antioxidant enzymes. It seems like that early life stress causes behavioral and cognitive deficits via an oxidative stress-mediated weakening of neuronal connections. The central hypothesis of this Dissertation is that the ability to acquire susceptibility or resistance to stress-induced behavioral and cognitive deficits resides in oxidative-antioxidative balance within the CNS. This balance is maintained by transcriptional and epigenetic mechanisms. Therefore, our long-term goal is to investigate a) the role of early life stress on behavior and cognition across different ages in rats, b) reveal resilience and susceptible phenotypes and c) to identify the role of oxidative mechanisms in the regulation of behavioral and cognitive function and resilience. We propose to utilize a comprehensive approach to address our goals. In Aim 1, the effect of induction of early life trauma was examined using a rat model of early-life stress on later life behaviors. Sprague Dawley (SD) rats were exposed to single prolonged stress (SPS) at postnatal day (PND) 25. Behavior tests to assess anxiety-like behavior, depression-like behavior, and learning and memory function were performed at different stages of development during PND 32, 60 and 90. Resilience and susceptibility phenotypes also were examined. In Aim 2 we examined the effect of early life stress on oxidative stress mechanisms as well as transcriptional and epigenetic regulation of specific genes that presumably control antioxidative capacity. We focused explicitly on Keap1-Nrf2 and NF-κB pathway. SD rats were exposed to SPS at PND25. One group of rats were sacrificed at PND32, the other group of rats was sacrificed at PND90. Blood was collected, and plasma was used to examine systemic markers of oxidative stress and physiological stress. Brains were harvested, and specific brain areas were isolated, and homogenates were prepared for conducting biochemical analysis to determine the effect of early life SPS on oxidative-antioxidant balance, and activation of redox-sensitive pathways such as Nrf2 and NF-κB pathways. Studies proposed in aim 1 revealed that rats exposed to SPS exhibited both anxiety- and depression-like behavior at PND32. Moreover, short-term (STM) but not long-term memory (LTM) was impaired. Rats exposed to SPS at PND60 exhibited anxiety- but not depression-like behavior. STM but not LTM was impaired. Rats exposed to SPS at PND90 exhibited fearful (as indicated by elevated plus maze test) but not an overall anxiety-like behavior (in light and dark test). These rats also displayed significant depression-like behavior with no changes in STM or LTM. Interestingly, when data was further analyzed, two subsets of PND90 rats exposed to SPS were identified, “susceptible”: with depression-like behavior and “resilient”: without depression-like behavior. Importantly, while resilient group expressed early signs of anxiety- (at PND32 and PND60) and depression-like behavior (at PND32), these behavioral deficits were absent at PND90. On the other hand, susceptible PND90 rats exposed to SPS expressed later onset of anxiety-like behavior (at PND60), while depression-like phenotype was evident only later on at PND90. At the biochemical level, SPS exposure at PND25 led to an increase in oxidative stress in specific regions of the brain (pre-frontal cortex), as indicated by the increased level of oxidative stress marker at PND32 and PND90. SPS exposure at PND25 also led to an initial increase in antioxidant enzyme expression at PND32 and a decrease in antioxidant enzyme expression at PND90. The increase in oxidative stress and the decrease in antioxidant enzymes at PND90 correlates with the depressive phenotypes in SPS rats at PND90. Further biochemical studies revealed a state of a compromised Nrf2 pathway and activated NF-κB pathway in the pre-frontal cortex (PFC) homogenates. The state of compromised Nrf2 pathway and activated NF-κB pathway was indicated by a decrease in the levels of Nrf2 and increased levels of NF-κB, as well as NF-κB-mediated increased levels of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) in PFC. In summary, our findings suggest that early life stress caused co-occurrence of anxiety and depression-like behavior at PND32 (mimics human early-adolescent period). This co-occurrence was lost at PND60 with a demonstration of anxiety- but not depression-like behavior. Later, depression but not the anxiety-like behavior was observed at PND90. It seems that behavioral adaptations occur at the critical PND60 stage (mimics human late-adolescent period) (Sengupta 2013), where behavioral and cognitive switching occurs, thereby, expressing susceptible and resilient phenotypes. Moreover, susceptible phenotype correlates with an increase in oxidative stress and a decrease in antioxidant enzymes in the emotion-regulating brain region of the PFC. The correlation between susceptible phenotype and increased oxidative stress markers suggests that the early life stress causes a buildup of oxidative stress, which negatively affects neuronal circuitry that contributes to depressive phenotypes. The increase in oxidative stress induced by early life stress activates NF-κB pathway, which triggers cellular inflammatory responses.Item Cell-Type-Specific Requirement of Progestereone Receptor for Suppression of Cell Proliferation and Carcinogenesis in the Cervix(2020-05) Park, Yuri; Chung, Sang-Hyuk; Bawa-Khalfe, Tasneem; Lin, Chin-Yo; Han, Sang JunCervical cancer is the third most leading cause of cancer deaths in women worldwide. Persistent infection with high–risk human papillomaviruses (HPVs) is causally associated with cervical cancer. High–risk HPV E6 and E7 oncoproteins promote cell proliferation by inactivating p53 and pRb cellular tumor suppressors, respectively, which accelerates cervical carcinogenesis. However, HPV is not sufficient for cervical cancer. Epidemiologic evidence suggests that females sex hormones contribute to cervical carcinogenesis. Consistently, chronic estrogen (E2) treatment promotes cervical carcinogenesis in HPV transgenic mouse models expressing HPV16–E6 and/or HPV16–E7. Our lab has demonstrated that synthetic progestin medroxyprogesterone acetate (MPA) prevents and regresses cervical cancer in the HPV transgenic mouse model. Our lab also has demonstrated that stromal estrogen receptor alpha (ERα) mainly mediates pro–tumorigenic action of E2. The goal of my study is to determine roles of epithelial and stromal progesterone receptor (PR) in suppression of cell proliferation and cervical cancer. In Chapter 1, I investigate whether deletion of PR promotes cervical carcinogenesis without chronic E2 treatment. I found that ablation of PR expression in the cervical epithelium sensitizes HPV transgenic mice to spontaneous cervical carcinogenesis. I also found that spontaneous cervical cancers mimic ERα and PR status in cervical cancer patients, which provides a platform to study ERα–negative and PR–negative cervical cancer. In Chapter 2, I determine roles of stromal and epithelial PR in regulation of cervical epithelial cell proliferation and survival in different E2 concentrations. I found that both epithelial and stromal PR are required for P4 to exert its anti–proliferative and pro–apoptotic actions regardless of E2 concentrations. Our lab has demonstrated that epithelial PR is required for cervical cancer regression by MPA. These observations suggest that stromal PR is also required for progestin therapy to work. In other words, MPA would be effective in treating cervical cancer expressing PR in both cancer and surrounding stroma. My findings further support that PR is a tumor suppressor in cervical cancer and provide a biomarker for patient selection for a potential clinical trial.Item Deciphering a Novel SUMO-dependent lncRNA Degradation Mechanism on Telomere during Cell Cycle Progression to Impact Aging and Cancer Onset(2021-05) Quttina, Maram Adnan Sami; Bawa-Khalfe, Tasneem; Lin, Chin-Yo; Dauwalder, Brigitte; Merchant, Fatima AzizThe exosome complex component EXOSC9 (Rrp45) is an essential subunit of the RNA degradation machinery in eukaryotic cells. In addition to its supportive role in exosome-based mRNA surveillance, EXOSC9 also exhibits independent catalytic activity to degrade select cytosolic mRNA and is present in the nucleus and chromatin fractions. Like mRNA, long non-protein-coding RNA (lncRNA) is indispensable for normal cell physiology and, consequently, tightly regulated in the cell. Yet, unlike mRNA, substantially less is known about the mechanisms for lncRNA degradation. It is important to delineate the regulatory control of lncRNA degradation, particularly telomeric repeat-containing RNA (TERRA), as the TERRA-telomere R-loops dictate cell cycle progression and genomic stability. Recent studies in our lab indicate that the SUMO-modified heterochromatin protein 1alpha (HP1α) more readily interacts with TERRA than unmodified HP1α. Interestingly, the SUMOylated form of this canonical chromatin remodeling protein also recruits EXOSC9 to the chromatin. During the S/G2 transition of the cell cycle, we observed an accumulation of SUMOylated HP1α, the interaction between HP1α- EXOSC9, and a significant TERRA loss. Hence, we expect that these events support normal cell cycle progression; specifically, chromatin-enriched EXOSC9 requires interaction with SUMO-modified HP1α to degrade TERRA and facilitate telomere replication. Results from this study will delineate for the first-time a cell-cycle driven mechanism for degradation/clearance of chromatin-bound lncRNA. Persistent activation of this lncRNA-clearance system with induction of a SUMO-mimetic HP1α causes accumulation of DNA damage at telomeres. Consistently in normal human cells, SUMO-modification of HP1α is tightly regulated via an active deSUMOylase SENP7 as demonstrated by us and others. The full-length SENP7 (SENP7L) binds and reduces SUMO post-translational modification of HP1α in multiple mouse and human cells including mammary epithelial cells. Unlike other deSUMOylases, SENP7L is enriched at heterochromatin sites and directs condensed chromatin architectures as defined in multiple cell models. While these studies establish a critical role of SENP7L, it is unclear if the enzymatic activity of SENP7L is required for its biological actions. Here, we used a genetically engineered mouse model (GEMM) first established in our lab to evaluate how catalytical activity of SENP7L directs HP1α modification and normal mouse mammary epithelial development. In normal mouse development, we report an induction of SENP7L during the pubertal stage, specifically in mammary hierarchical stem/progenitor cells (MaSC) and the MaSC niche called the terminal end buds. The knockdown of SENP7 catalytic activity in this GEMM increased MG's branching morphogenesis and accelerated differentiation in an ex vivo organoid system. Concurrently, sustained SUMOylated HP1α is enriched in chromatin fraction of this SENP7 dead GEMM. Recent studies in our lab indicate an analogous induction of SUMOylated HP1α with loss of SENP7L in endocrine-resistant hormone receptor positive breast cancer (BCa); this BCa subtype is a therapeutic challenge as patients are unresponsive to conventional endocrine-targeting drugs and readily progress to metastatic disease. Hence, we expect our research could present novel biomarkers to identify endocrine non-responders and identify alternative therapeutic strategies for these BCa patients.Item Developmental Malformations in Zebrafish Caused by Exposure to Environmental Pollutants(2018-12) Thrikawala, Savini Upara 1986-; Gustafsson, Jan-Åke; Bondesson, Maria; Zhang, Xiaoliu Shaun; Bawa-Khalfe, Tasneem; Frigo, Daniel E.; Wagner, DanielIt is estimated that there are about 80,000 man-made chemicals released to the environment with little to no toxicity information. Exposure to these chemicals is identified as one of the risk factors for causing birth defects. We used zebrafish as a model to identify and predict environmental chemicals causing congenital malformations. High-throughput screening of 292 unique chemicals from the ToxCast Phase I library identified 38 skeletal disruptor compounds (SDCs) producing bone and craniofacial cartilage malformations in zebrafish. We used ToxCast in vitro data and RT-qPCR to predict that these compounds affect vitamin D metabolism and dopamine transporters resulting skeletal malformations. Transcriptomics data identified that exposure to cyproconazole, a prominent SDC, induce adipogenesis, while repressing osteo and chondrogenesis. Next, we investigated vascular disruptor compounds (VDCs) that cause malformations in vivo in zebrafish vasculature and in vitro in human umbilical cord endothelial cells (HUVECs). ToxCast data correlations identified that VDCs alter signaling of nuclear receptors like ER, AR and transcription factors like Hif1α, causing vascular perturbations. The ToxCast in vitro assays that correlated with either the identified SDCs or VDCs were used to predict chemicals with in vivo toxicity in zebrafish using the ToxPi tool. Exposures to predicted SDCs and VDCs were found to induce bone and vascular malformations, respectively. We confirmed that chemicals predicted to not have any negative effect on the skeleton or vasculature were indeed inert in zebrafish. The predicted VDCs affected in vitro HUVEC tube formation, while predicted non-VDCs did not. Lastly, we investigated the effects of a widely used insecticide, pyriproxyfen, which is suspected to cause microcephaly. We identified that pyriproxyfen exposure causes significantly smaller heads, smaller midbrains and a prominent gap along the mid-dorsal margin in the brains in zebrafish compared to controls. Transcriptomic data proposed that pyriproxyfen affects neuronal and axonal migration and cerebrovascular development. In conclusion, we used zebrafish embryo toxicity in combination with pathway-based in vitro data to identify environmental chemicals that cause developmental malformations and their mechanisms of action. Most of the identified molecular mechanisms are conserved among vertebrates, suggesting that these compounds could potentially be harmful to humans and other mammals.Item Discovering Novel RNA Regions to Develop Biomarkers and Therapeutic Opportunities for Breast Cancer & SARS-CoV-2(2020-12) Mistretta, Brandon; Gunaratne, Preethi H.; Bawa-Khalfe, Tasneem; Bedrosian, Isabelle; Briggs, James M.; Zhang, Xiaoliu ShaunBreast cancer accounts for 23% of all cancer deaths and second most related mortality in women. Recently, advances in the field of immuno-oncology have demonstrated therapeutic effects of boosting endogenous T cells to combat human cancers. Fusion junctions in chimeric RNAs provide potential neoantigen peptide regions expand the potential repertoire of targets for therapeutic vaccines. Here we focused on extracting neoantigens from fusion transcripts identified from RNA-sequencing of breast tumors. We present 20 novel fusion transcripts from 75 patients (TNBC, HER2+, and HR+), which are not present in normal breast. We focused on the NSF [Exon 1-12]-LRRC37A3 [Exon 5-13] fusion detected in 24% of the tumor samples. Major ORFs predicted including NSF-Exon 1-12-KFPRKLYFLH (C-terminal truncation) and MISNQ-LRRC37A3 Exon 5-15 (N-terminal truncation) were analyzed through MHC Class I binding predictor (MHCnuggets). A total of 15 different 8-11 mer neoantigen peptides discovered from the NSF and LRRC37A3 truncations were predicted to bind to a total of 35 unique MHC class I alleles with a binding affinity of IC50<500nM. We present here a proof of principle strategy to identify immunogenic neoantigen candidates from fusion transcripts that can serve as reagents for developing tumor vaccines for the prevention and treatment of breast cancer. The COVID-19 pandemic has resulted in > 54.7 M cases and > 1.32 M deaths as of November 16, 2020, due to systemic inflammation and multiorgan dysfunction. We performed Whole Genome Sequencing (WGS) of SARS-CoV-2 RNA genome on 30 patients and found a 40% false negative rate from the FDA N1|N2 qPCR test. WGS reads analyzed through the CLC Genomic Pipeline revealed ‘degradation resistant regions’ from the SARS-CoV-2 genome, which we used to design new primer pairs to significantly improve the sensitivity and specificity of detection. Adding primer sets that capture the host response in relation to cytokine storm, cardiac, and vascular endothelial dysfunction we constructed a combined ‘Viral Infection|Host Response Detection’ panel that could be used to accurately identify asymptomatic super-spreaders and triage patients who are likely to develop severe symptoms from SARS-CoV-2 infections. The goal was to establish a clinical decision making framework to manage the pandemic under limited hospital capacity.Item Functional Regulation of Integrin Affinity by Divalent Cations, Glycosylation and Simvastatin(2021-12) Moussa, Zeinab; Şen, Mehmet; Briggs, James M.; Dryer, Stuart E.; Bawa-Khalfe, Tasneem; Ma, QingIntegrins are heterodimeric metalloproteins cell surface receptors that are expressed on every nu- cleated cell in the body. Integrin dysfunctions are implicated in many diseases—including bleeding disorders, inflammation, and cancer. Thus integrins are critical and proven drug targets. Integrins are unique in their bidirectional signaling and the transmission of the signal between the α and β subunits. β2 integrins are composed of a variable α (αL, αM, αX, αD) and a constant β2 subunit and are exclusively expressed on leukocytes. The α subunit contains an inserted I domain with a metal- dependent adhesion site (MIDAS) that acts as a ligand-binding site thereby defining the functional property of the integrin. Divalent cations are indispensable for integrin functions, stabilizing the integrin structure and regulate integrin-ligand interaction. In this study, the effect of divalent cations on integrin function was investigated for both αXβ2 and αDβ2 integrins. Locking the β2 I-domain into the binding-incompetent state using either inhibitory monoclonal mAb, TS1/18, or small-molecule inhibitor XVA143 enabled us to assess the effect of the divalent cations on the αI- domain. The tested divalent cations including Mg2+, Co2+, Cd2+, and Ni2+ induced an increase in the αI-domain affinity towards physiological ligands iC3b and fibrinogen. In this study, the effect of individual N-glycans on αXβ2 expression, conformation and affinity was investigated. This study suggests that individual N-glycan sites exhibit different roles on the affinity of the αXβ2, emphasizing the importance of studying the functional role of individual N-glycans in integrin-ligand affinity and conformation. Better understanding integrin conformation and ligand affinity could lead to improved ther- apeutics for the treatment of inflammatory diseases. In this study, we identify simvastatin as a competitive antagonist of αXβ2. Optimizing full length integrin purification is critical to investi- gate the structural and functional regulation of integrins in the presence of small molecules such as simvastatin. The cloning and purification strategies developed can be applied for that purpose.Item Impact of SUMO Post-translational Modification on Breast Cancer Development(2023-04-13) Babajide, Funmi; Karami, Samaneh; Waiters, Kacie; Peidl, AnthonyProteins are subject to post-translational modifications (PTM) that ensure the plethora of unique cellular functions. SUMO-PTM allows proteins to maintain cellular homeostasis and respond to abnormal stressors. SUMO-PTM of proteins is a dynamic reversible process important to cellular biology and normal human physiology. Consistently an imbalance of SUMO-modified proteins (or SUMOylation) can lead to breast cancer development and support onset of metastatic disease. Hence, the long-term objective of our studies is to understand and develop appropriate therapeutics to restore SUMO-PTM balance in breast cancer. At a molecular level, the Small Ubiquitin-like Modifier (SUMO) molecule forms a covalent bond with lysine residues on target proteins with the work of SUMO-specific family of conjugating enzymes including SUMO E3 ligases. Inversely, SUMO-specific protease or deSUMOylase hydrolyze the bond between SUMO and the target protein to maintain the unmodified form of the substrate. Here, we examine protein interactions between novel SUMO E3 ligase and its target protein in breast cancer cells stressed with conventional anti-cancer therapy. Concurrently, we test how excessive SUMO-PTM impacts breast cancer development using a novel genetically engineered mouse model (GEMM). These results can aid in the further understanding of protein function and cellular homeostasis in context of breast cancer development and resistance to conventional anti-cancer therapy.Item Mechanism of Dopamine 2-like Receptor Signaling in The Blood Brain Barrier that Regulates Male Courtship in Drosophila Melanogaster(2023-12) Gautam, Sumit; Dauwalder, Brigitte; Gunaratne, Preethi H.; Bawa-Khalfe, Tasneem; Bond, Richard A.In Drosophila melanogaster, courtship behavior and the brain circuits that mediate it are well characterized. Blood Brain Barrier (BBB) is an important part of the brain. It is purely glial in Drosophila and produces sex-specific modulators of male courtship behavior. One of the sex-specific BBB transcripts encodes a dopamine receptor, D2R, a G protein coupled receptor. D2R plays a major role in many central nervous system functions in both invertebrates and vertebrates. In Drosophila, we have previously found that D2R is preferentially expressed in the SPG cells of the adult male BBB and plays an important role in the regulation of male courtship. However, the molecular pathways downstream of D2R that modulate courtship behavior were unknown. D2R is a member of the family of G protein-coupled receptors (GPCRs) that can signal through two transducers: G proteins and β-arrestin. This study aims to characterize the role of G protein or arrestin signaling of D2R in the BBB for proper male courtship. The question was addressed by employing a genetic and a pharmacological approach. Two unique molecular genetic tools were generated by site-directed mutagenesis, a G protein-biased and a β-arrestin-biased version of D2R. These receptor versions, which can preferentially activate either the G protein or the β-arrestin downstream pathways were examined for their ability to rescue the courtship defects of D2R mutants. In a complementary pharmacological approach, well-known synthetic agonists, the G protein-biased agonist, MLS1547 and the β-arrestin-biased agonist, UNC9994 were administered to flies to examine their potential to rescue the courtship defects of D2R hypomorphic flies. The results from the genetic approach demonstrate that arrestin-mediated, but not G protein-mediated, signaling downstream of D2R is essential for normal male courtship. In agreement with this finding, the courtship defect of D2R hypomorphic flies was rescued by the administration of the arrestin-biased agonist, UNC9994. Together, these findings demonstrate that arrestin-mediated signaling through the D2R receptor in the BBB is required for normal male courtship behavior.Item Novel Targets And Therapeutics in Pancreatic Ductal Adenocarcinoma(2017-07-31) Karaboga, Husna 1986-; Lin, Chin-Yo; Bailey, Jennifer M.; Chung, Sang-Hyuk; Bawa-Khalfe, Tasneem; Zhang, WeihuaPancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all major malignancies. As there is no effective treatment method, it is emerging as one of the most deadly cancers. Oncogenic activation of KRAS is the most commonly seen mutation with 95% frequency in PDAC. Inhibition or down-regulation of KRAS activity impairs the pancreatic cancer cell metabolism and survival. However, directly targeting mutant KRAS has largely failed. There are numerous attempts to uncover novel targets and therapeutics generating potent response in PDAC treatment. Revealing molecular and genetic underpinnings of PDAC is essential to find effective targeting mechanisms and treatment options. The aim of this dissertation is to characterize new PDAC associated molecules (Chapter 2) and potential therapeutics (Chapter 3). In Chapter 2, we focused our interest in long non-coding RNAs (lncRNAs), which are expressed from nearly one fourth of the total encoded genes in the human genome. Generally, their expression is lower than protein-coding genes; they are strikingly tissue specific and have roles in various concepts of cell biology. We showed abnormal expression and dysregulation of lncRNAs are associated with PDAC and characterized a potentially important novel lncRNA, PANCRNA1, in PDAC formation and progression. We also elucidated the functional role of PANCRNA1 as a cancer driven molecule and determined its expression status depends on KRAS oncogenic mutation. This study shows first time the expression and function of PANCRNA1 in PDAC. Liver X receptor β (LXRβ) has been emerging as a promising therapeutic target in PDAC. It mainly regulates lipid metabolism and cholesterol homeostasis, which are dysregulated in cancer. Targeting LXRβ show anti-tumorigenic effect in PDAC. However, there is no LXRβ-specific ligand designed to use in cancer studies. In Chapter 3, we screened novel LXRβ ligands and reported two ligands that showed significantly greater antigrowth effects on PDAC than currently used synthetic ligands. Our findings provide mechanistic insights on identified ligands in lipid metabolism and also demonstrated the effect of KRAS mutation on treatment outcome. These compounds have the potential to revolutionize the study and treatment of pancreatic cancer and other deadly cancers that currently lack effective treatment options.Item Novel Tumor Suppressive Mechanisms of Estrogen Receptor β in Prostate Cancer(2020-05) Chaurasiya, Surendra P. 1976-; Gustafsson, Jan-Åke; Weigel, Nancy L.; Ström, Anders M.; Lin, Chin-Yo; Bawa-Khalfe, TasneemEstrogen receptor β (ERβ) was first identified in the rodent prostate and is abundantly expressed in human and rodent prostate epithelium, stroma, immune cells, and endothelium of the blood vessels. Genomic deletion of ERβ led to hyperplasia of prostate epithelium as well as upregulation of androgen receptor (AR) regulated genes. ERβ has been shown to inhibit proliferation and induce apoptosis in prostate cancer cells; however, role of ERβ in regulating AR activity in prostate cancer has not been studied in detail. Additionally, the role of ERβ in PI3K/Akt/PTEN pathway, which is one of the most altered in prostate cancer, is not known. Chapter 2 of this dissertation describes the role of ERβ in regulating PI3K/Akt/PTEN pathway. ERβ upregulated INPP4B in prostate cancer cells, PC3, as well as non-malignant cells BPH-1. Upregulation of INPP4B inhibited Akt activity measured by phosphorylation of Ser473 and its downstream target GSK3β. Further, we show that ERβ inhibited migration of PC3 cells by upregulating INPP4B in wound healing assays. This regulation may indicate a role for ERβ in metastasis suppression. Androgen receptor is the main driver of primary as well as metastatic prostate cancer. Chapter 3 describes the role of ERβ in regulating AR expression and activity in prostate cancer cells LNCaP. Using global transcriptomic analysis of ERβ- expressing LNCaP cells, we found AR-signaling is the most downregulated effect after ERβ activation. We validated this regulation independently by transcript and protein measurement of established AR target genes FKBP5, CaMKK2, TBC1D4, as well as by luciferase reporter assay. We further demonstrated that downregulation of CaMKK2 inhibits activity of AMPK, a major energy sensing mechanism in cells. Taken together, these findings support tumor suppressive effects of ERβ in prostate cancer through novel mechanisms and indicate possibilities for therapeutic intervention.Item Pipeline for the Zebrafish Model as a Predictor of Specific Teratogenic Effects(2020-05) Mirchandani, Rachna; Gustafsson, Jan-Åke; Bondesson, Maria; Schwartz, Robert J.; Bawa-Khalfe, Tasneem; Eisenhoffer, George T.Over 80,000 synthetic chemicals are currently in commercial use without a full assessment of their adverse effects, especially on developmental stages of life. Exposure to some of these chemicals has been linked to birth defects, chronic disease and cancer. Synthetic chemicals are also present in everyday items, such as food, household cleaners and lawn care products. Zebrafish (Danio rerio) are an effective alternative in vivo model to standard mammalian models for teratogenic studies owing to their rapid development, low cost, and genetic homology to humans. The availability of transgenic fish that express fluorescence in specific tissues or organs allows for detection of tissue-specific structural malformations in live fish. We have used the zebrafish model to understand the adverse effects that potential teratogens have on living systems, and to establish a rapid and reliable screening method in vivo. Transgenic fish were used to screen for chemicals that perturb angiogenesis and posterior lateral line (PLL) development, and the screening results subjected to computational toxicology to elucidate the molecular pathways by which teratogens cause these specific phenotypic effects. We screened 175 ToxCast Phase I chemicals in zebrafish for vascular teratogenicity (vascular disrupting chemicals; VDCs) and identified 10 chemicals that interfered with intersegmental vessel (ISV) development. For PLL teratogenicity (PLL disruptors, PLLDs), we tested all 292 chemicals from the ToxCast Phase I library and identified 22 chemicals that reduced the number of deposited neuromasts. The chemical hit lists for each of the developmental perturbations observed were then compared to the ToxCast in vitro assays to produce a phenotype-specific assay profile of genes or molecular pathways through which these chemicals may be acting. The assay profiles were used to predict other chemicals from the ToxCast Phase II library that could be potential VDCs or PLLDs. In summary, we have established a rapid screening method for phenotypic outcomes in zebrafish and combined it with computational analysis of ToxCast in vitro data to obtain phenotype-specific assay profiles that can further be used to rank toxicants tested in the Tox21 project to speed up prioritization of chemicals for further testing in vivo, and in the long run result in an improved risk assessment for human and environmental health.Item Proteomic Analysis of Post-Translational Modifications and Signaling Pathways in Pancreatic Cancer(2019-12) Vo, Hiep T. 1990-; Gao, Xiaolian; Bawa-Khalfe, Tasneem; Frigo, Daniel E.; Gunaratne, Preethi H.Signaling pathways mediated by receptor tyrosine kinases (RTKs) and protein kinases play essential roles in cellular physiology. Src Homology 2 (SH2) domains bind to specific phosphotyrosine motifs and link activated RTKs to several downstream pathways. Studying SH2 domain-containing proteins implicated in cancer signaling networks under given therapies can yield significant insight into the molecular basis of drug responses, thus having valuable clinical applications. Here, we demonstrate a microfluidic, peptide-microarray biochip technology (μPepArray™) for the label-free and multiplex detection of the endogenous SH2 domain proteins and signaling pathways from total protein lysate. The biochip contains 3968 reaction chambers, where the immobilized phosphopeptides serve as the molecular probes to capture present SH2 domain proteins. Using pancreatic cancer cells treated with epidermal growth factor receptor inhibitors (EGFRis), we identified the differentially expressed SH2 domain proteins such as SHP2, PLCG1, PI3K, and therefore the downstream phosphotyrosine-mediated signaling events occurred in response to treatments. Our findings revealed a signaling compensation between the EGFR/PI3K/Akt and mTORC2/PKCα/ERK pathways as a mechanism of EGFRi responses and potential resistance in BxPC-3 cells. We propose that the proteomic information provided by μPepArray™ technology could hold clinical significance by assisting in the cancer biomarker identification and guiding future treatment decisions. In the second approach of this dissertation, we utilize μPepArray™ technology for the high-throughput identification of histone SUMOylation. SUMOylation is a dynamic post-translational modification (PTM) process which covalently attaches a small ubiquitin-like modifier (SUMO) to a protein substrate. This modification alters protein functions and activities, ultimately affecting cellular responses and homeostasis. Histones play essential roles in epigenetic regulations. Different histone PTMs directly dictate chromatin structure and transcriptional activities. Identifying histone SUMOylation and specific lysine residues that are SUMOylated has been challenging due to limitations in technology. Using a customized library of lysine peptides derived from histone sequences, we have identified multiple histone SUMO modification sites as well as peptides that bind non-covalently to SUMO on the microarray chip. We propose that μPepArray™ technology offer a rapid screening assay to determine protein post-translational modifications, which will provide significant insights into PTM substrate specificity and their functions.Item Serial Knockout of Diphthamide Biosynthesis (DPH) Genes Result in the Secretion of Immuntoxins and Leads to the Enhancement of Pseudomonas Aeruginosa Exotoxin A (PE) Based Immuntoxin(2019-12) Atkins, Jason 1979-; Zhang, Xiaoliu Shaun; Frigo, Daniel E.; Lin, Chin-Yo; Bawa-Khalfe, Tasneem; Varadarajan, NavinDiphthamide is a post-translational modification of histidine found on eukaryotic elongation factor 2 (eEF-2), which is generated in a multi-step biosynthetic process by a series of seven diphthamide biosynthesis enzymes (DPH1-7) and is highly conserved among eukaryotes and archaebacteria. Previous studies have shown that inactivation of DPH1, 2, 4, or 5 genes lead to the prevention of ADP ribosylation of diphthamide and resistance to diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). However, the physiological function of diphthamide has remained rather obscure. We have used CRISPR-Cas9 to conduct systemic knockout (KO) of all the seven DPH genes in multiple cell lines and have investigated the long-term effect on cellular survival and protein synthesis in both in vitro and in vivo characterizations. Our data show that, in contrast to early reports, the deletion of DPH1, 2, 4, or 5 has either an immediate or delayed detrimental effect on cell proliferation, survival and protein synthesis. Most interestingly, cells can completely tolerate the deletion of either DPH6 or DPH7 with or without the presence of DT or PE toxin. We also conducted a series of combinatorial KOs of DPH genes and the data are consistent with the notion that loss of any of DPH genes results in the phenotype of the first KO gene in consecutive order. These observations thus shed new light on the distinct functions of these diphthamide-eEF2 conversion enzymes and also lead to the conclusion that only DPH 6 and 7 can be inactivated without affecting cell viability.Item Strategies to Identify and Overcome the Deregulated SUMO-Modification in Endocrine Resistant Breast Cancer(2020-05) Bahnassy, Shaymaa; Bawa-Khalfe, Tasneem; Weigel, Nancy L.; Chen, Li; Chung, Sang-Hyuk; Frigo, Daniel E.Resistance to canonical endocrine therapy (ET) remains a major therapeutic challenge for patients of the frequently diagnosed hormone receptor positive (HR+) breast cancer (BCa) subtype. Previous studies show that deregulated Small Ubiquitin Like Modifier (SUMO)-post-translational modification (PTM) of proteins drive breast tumorigenesis. However, if and how the SUMO-proteome changes with chronic treatments of ET is unknown. A collective series of studies demonstrate that androgen receptor (AR) supports resistance to ET and correlates with BCa metastasis. The AR is also a well-known target for SUMO-PTM. Yet, whether AR SUMOylation contributes to the metastatic phenotype of recurrent ET-R BCa remains undefined. Here, we report concurrent accumulation of global SUMOylome and SUMOylated AR in acquired and intrinsic ET-R hormone receptor-positive (HR+) BCa lines. We identified heat shock protein (Hsp27) as a novel SUMO-E3 ligase for AR. Ligand-independent SUMO-AR is chromatin-bound, transcriptionally active, and favors the expression of epithelial-mesenchymal transition genes. Our results show that concurrent targeting of unmodified and SUMO-modified AR is required to attenuate the migration and metastatic phenotype of ET-R BCa. Collectively, we propose increased global SUMOylation and SUMO-modified AR to stratify BCa patients that would benefit from combinatorial treatments of AR antagonists and SUMO inhibitor therapy. High-throughput approaches for rapid screening of SUMO-PTMs are lacking. Also, non-canonical SUMO-acceptor sites and SUMO-interacting motifs (SIM) are a particular hurdle for conventional, laborious biochemical approaches. Here, we apply a microfluidic, peptide-microarray biochip technology (μPepArray™) to rapidly screen for non-covalent and covalent SUMO sites for two challenging protein substrates Hsp27 and core histones, respectively. Using an unbiased screening approach, we identified and confirmed multiple functional SIMs on an established SUMO-E3 ligase Hsp27. Additionally, we designed a unique peptide array to identify several new SUMOylation sites on each of the four core histones. Collectively, we propose μPepArray™ as a rapid screening tool for identifying various forms of SUMO-PTM, applicable for studying SUMO-regulated cellular processes in health and disease.Item Suppression of Cervical Cancer by the Progesterone Receptor Signaling(2016-05) Mehta, Fabiola Melissa 1985-; Chung, Sang-Hyuk; Bawa-Khalfe, Tasneem; Frigo, Daniel E.; Weigel, Nancy L.Cervical cancer is the fourth-most common cancer in woman and fourth leading cause of cancer death worldwide. The majority of cervical cancer is associated with high-risk human papillomaviruses (HPVs). Their tumorigenic potential stems mainly from viral oncoproteins E6 and E7, which are best known to inactivate p53 and pRb tumor suppressor, respectively. Epidemiological evidence suggests that, in addition to persistent HPV infections, other cofactors are required for cervical cancer. Multiple pregnancies and oral contraceptive use increases the risk for cervical cancer in HPV-infected women, implicating a role of estrogen and progesterone in cervical cancer. Prior studies utilizing an HPV transgenic mouse model expressing E6 and E7 (K14E6/K14E7) have demonstrated the requirement of estrogen receptor alpha (ERα) and estradiol (E2) for cervical carcinogenesis. Accumulating evidence suggests that ERα may be important for human cervical cancer. The role of progesterone and progesterone receptor (PR) in cervical cancer remains elusive. Our laboratory has demonstrated that PR agonist medroxyprogesterone acetate (MPA) promotes regression of cervical cancer and cervical intraepithelial neoplasia (CIN) in the K14E6/K14E7 mice. Goals of my dissertation project were to determine whether cervical cancer recurs after MPA therapy and whether epithelial PR is required for therapeutic effect of MPA. Using the K14E6/K14E7 mice, I found that cervical cancer recurred even if MPA treatment was continued, and recurring cervical cancer expressed PR but was refractory to MPA. In addition to PR, MPA interacts with other nuclear receptors including glucocorticoid receptor. Using the Cre/LoxP recombination system, I found that epithelial PR promoted apoptosis and inhibited proliferation in the cervical epithelium. I also determined that epithelial PR was required for MPA-mediated regression of cervical cancer, which was inhibited by E2. My results strongly support the hypothesis that epithelial PR is ligand-dependent tumor suppressor in cervical cancer. Approximately 33% of cervical cancer expresses PR. My results suggest that PR expression may not be sufficient to benefit from MPA therapy. My results also warrants further study to determine mechanism of recurrence and therapy resistance, which will facilitate the development of a better therapy for the disease.Item The Pharmacology and Molecular Mechanisms of ADGRF1 (GPR110) in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer(2022-12) Abdulkareem, Noor; Trivedi, Meghna V.; Hussain, Tahir; Kim, Hee-Yong; Bawa-Khalfe, Tasneem; Liu, XinliHuman epidermal growth factor receptor-2-positive (HER2+) is overexpressed in 15-20% of breast cancer (BC) patients. Despite the success of the combination regimen containing multiple anti-HER2 drugs plus chemotherapy, one-third of patients develop drug resistance and hence progressive disease eventually responsible for early mortality. On the other hand, one-third of patients with HER2+ BC have pathological complete response, a surrogate for survival, to chemotherapy-sparing anti-HER2 drug combinations that have lower toxicities. Therefore, new drug targets that improve efficacy of anti-HER2 drugs and/or chemotherapy will help to prolong survival and avoid toxicities. G protein-coupled receptors (GPCRs) are known to be excellent drug targets due to their plasma membrane localization, high specificity and target-selectivity of ligands, and availability of large GPCR-targeting chemical libraries for screening. Over 30% of the Food and Drug Administration (FDA)-approved drugs target GPCRs and are used to treat a wide-range of chronic diseases, underscoring their overall favorable long-term safety profile. Our laboratory has previously identified ADGRF1 (formerly known as GPR110), an understudied and druggable adhesion GPCR (aGPCR), to play a critical role in promoting tumorigenesis in HER2+ BC. We recently reported that ADGRF1 overexpression predicted poor survival in HER2+ BC patients. Also, ADGRF1 overexpression promoted, whereas its knockdown inhibited, the tumorigenic behavior in HER2+ BC cells. Similarly, ADGRF1-overexpressing HER2+ cells led to faster tumor initiation and growth in vivo. Conversely, when ADGRF1-overexpressing cells were injected with growth factor reduced Matrigel (1:1), the tumor formation and growth reduced dramatically. These data led us to speculate an interaction between ADGRF1 and extracellular matrix (ECM) components of Matrigel. In these studies, we aimed to (1) investigate ADGRF1 signaling pathways in mediating its effects on tumorigenesis, (2) evaluate the crosstalk of ADGRF1 with HER1/HER2 pathways and the role of STAT3 as a downstream mediator of tumorigenesis promoted by ADGRF1 signaling, and (3) identify the proteogenomic signaling network of ADGRF1, in HER2+ BC. Our studies presented here indicate a pro-tumorigenic function of ADGRF1 via coupling to Gαs and activation of STAT3 signaling. We show that ADGRF1 overexpression promotes quiescence and chemoresistance in HER2+ BC, further supporting its role in tumorigenesis. We also show that ADGRF1 switches from tumor promoting to tumor suppressive function likely upon interaction with laminin-111. ADGRF1 interaction with ECM protein(s) causes inhibition of Gαs coupling and STAT3 phosphorylation as well as increase in senescence. ADGRF1, upon interaction with ECM protein(s), also induces HER2 expression and enhances anti-HER2 drugs sensitivity in HER2+ BC. Insights into how ADGRF1 signaling is governed by laminin-111 will help to develop novel therapeutic strategies relevant to this interaction in HER2+ BC. ADGRF1 is an understudied aGPCR yet implicated in different cancers and diseases. Hence, our studies serve as the basis to explore ADGRF1 as a novel drug target in cancer and other diseases.Item The Role of Androgen Receptor Modification in the Development of Drug-Resistant Breast Cancer(2018-10-18) Mousa, VictoriaThe most common targeted drug treatment option for pre-menopausal women with ER+ breast cancer (BCa) is Tamoxifen. Roughly 70% of women diagnosed with breast cancer are diagnosed with ER+ breast cancer, nearly half of which develop resistance rendering treatment ineffective and often leading to metastasis of the cancer and subsequent mortality. For patients who develop Tamoxifen resistance (TamR), other treatment options are limited to highly toxic non-targeted chemotherapy. Our long-term objective is to delineate targetable pathways that drive resistance to Tamoxifen in hormone positive BCa. Tamoxifen resistant ER+ BCa cells exhibit increased levels of global SUMO 2/3 conjugation; SUMO post-translational modification directs protein function and stability. The androgen receptor (AR) is a target for SUMO-modification in TamR BCa and SUMOylation of the AR supports hyper-activation of AR and metastatic nature of the TamR cells. While we know AR SUMOylation favors TamR cell proliferation, it is unknown if AR SUMOylation supports the development of resistance to the drug Tamoxifen. We predict that AR SUMOylation supports the development of resistance to the drug Tamoxifen. We are investigating, using western blots, when the SUMOylation of the AR occurs upon treatment of Tamoxifen-sensitive ER+ BCa cells. Additionally, mRNA quantification will be performed to assess SUMO-regulating enzymes. We are also investigating if treatment with combination of SUMO inhibitor and AR antagonists prevent resistance to tamoxifen and development of more drug resistant cancer spheroids.Item The Role of Estrogen Receptor α in the Development and Maintenance of Uterine Epithelium in Mice(2020-12) Unsal, Esra; Chung, Sang-Hyuk; Bawa-Khalfe, Tasneem; Han, Sang Jun; McKeon, Frank D.The uterus is composed of uterine cervix and uterine body. The cervical lumen is lined by the K14+/p63+/K8- squamous and K14-/p63-/K8+ columnar epithelium, which are demarcated at the squamocolumnar junction (SCJ). Location of SCJ changes depending on several factors, including ovarian hormones, vaginal acidity, and age in women. The epithelial region that the SCJ moves is called the transformation zone (TZ), where most cervical cancers occur. In K14E7 mice expressing HPV16 E7, SCJ shifts towards the uterine epithelium in an estrogen/ERα-dependent manner. We observed that SCJ moves toward the ectocervical epithelium in estrogen-depleted ovariectomized mice. (i.e., retraction of squamous epithelium). Estrogen administration expanded the squamous epithelium. These results suggest that HPV and estrogen/ERα promote squamous differentiation in the cervix. To better understand the role of ERα in this process, I first characterized genesis of cervical squamous epithelium and SCJ in postnatal mice. At birth, the entire cervical epithelium was composed of K14-/p63-/K8+ cells. Several K14+/p63+/K8- epithelial cells appeared underneath K14-/p63-/K8+ epithelial cells in the endocervix at postnatal day 6 (P6). They expanded to the posterior part of the cervix as aging. At P16 onwards, the endocervical epithelium became stratified. The location and epithelial marker expression of SCJ at P16 was similar to that in ovariectomized adults, indicating that squamous epithelium development was complete at P16. ERα expression pattern in the cervical epithelium at postnatal age coincided with expansion of K14+/p63+ cells. To study the role of epithelial ERα in squamous differentiation and SCJ formation, I used Wnt7aCre/Esr1f/f mice lacking expression of ERα specifically in the epithelium of the female reproductive tract including the cervix-they will be referred to as ER epithelium deletion (ED). Interestingly, ED mice had a delay in formation of SCJ. The location of SCJ in both ED and ERα knockout (Esr1-/-) mice at prepubertal age (P21) was similar to that in wild-type mice. However, junction location was impaired in ED but not in Esr1-/- mice at adult age, suggesting that balance between epithelial and stromal ERα is required for maintenance of SCJ at adult age. I found that, in epithelial ERα-deficient ED mice, K14+/p63+/K8- basal cells were present underneath the columnar epithelium in the entire uterus at adult age. I found that, although rare, these cells were also present in wild-type mice as early as postnatal day 14. Uterine basal cells expressed proteins that are not expressed in the other epithelial cell types in the cervix and uterine body. I found that K14 and p63 were expressed in human endometrium at the mRNA level. I did cell lineage tracing experiments in Krt5CreER/+/ROSA26mTmG/+ mice and found that uterine basal cells contribute to the columnar epithelium. Traced cells were mostly in the glandular epithelium. My results showed that the number of glands decreased in in ED mice but not in Esr1-/- mice. My results suggest that the balance between epithelial and stromal ERα signaling is crucial for the development and maintenance of cervical SCJ and uterine glandular genesis.