Browsing by Author "Zhang, Weihua"
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Item Altered Glucose Metabolism in Cancer Metastasis and Drug Resistance(2013-08) Chen, Jinyu 1985-; Zhang, Weihua; Warner, Margaret; Khurana, Seema; Lev, Dina Chelouche; Gan, BoyiGlucose, one of the most important energy sources for living organisms, is first broken down through glycolysis then either undergoes oxidative phosphorylation in the mitochondrion or fermentation in the cytosol. Abnormal glucose metabolism was first discovered by Dr. Otto Warburg, i.e. cancer cells carry out irreversible fermentation of glucose in the presence of oxygen, which is also termed aerobic glycolysis. My studies focus on understanding altered glucose metabolism in cancer metastasis and drug resistance. We found that breast cancer brain metastasized cells developed enhanced oxidation of certain amino acids and gluconeogenic activity for survival and proliferation when glucose level is limited. We also found that drug-resistant colon cancer cells exhibited up-regulated aerobic glycolysis to meet the need of a higher amount of intracellular ATP to cope with chemotherapeutic stress. These results suggest alterations in glucose metabolism play critical roles in the development of cancer brain metastasis and drug resistance. The molecular mechanisms identified by this study may serve as potential therapeutic targets for cancer treatment.Item Characterization of Novel Liver X Receptor Ligands in Pancreatic Cancer(2020-12) Srivastava, Shivangi; Lin, Chin-Yo; Zhang, Weihua; Widger, William R.; Şen, Mehmet; Bailey, Jennifer M.Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer with a high mortality rate due to the lack of early detection and effective treatment options for advanced diseases. Efforts to directly target mutant KRAS found in >94% of PDACs have not been successful due to structural constraints. An alternative strategy to directly targeting KRAS is to identify and target druggable receptors involved in dysregulated cancer hallmarks such as metabolic reprogramming downstream of KRAS dysregulation. Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-modulated transcription factors and are involved in the regulation of genes which function in key cancer-related processes, including cholesterol transport, lipid and glucose metabolism, and inflammatory and immune responses. Modulation of LXRs via small molecule ligands has emerged as a promising approach in cancer therapeutics. Analysis of transcriptomic data from PDAC clinical samples reveals upregulation of LXR and its target genes in tumors. In a screen of a focused library of drug-like small molecules predicted to dock in the ligand-binding pocket of LXRβ, we identified two novel LXR ligands with more potent anti-tumor activity than current LXR agonists used in our published studies. Characterization of the two lead compounds (GAC0001E5 and GAC0003A4) indicates that they function as LXR inverse agonists which inhibit their transcriptional activity. Prolonged treatments with novel ligands further revealed their function as LXR “degraders” which significantly reduced LXR protein levels in all three PDAC cell lines tested. Using metabolomics approach, we discovered that GAC0001E5 inhibits glutamine anaplerosis and induces oxidative stress in PDAC cells. Furthermore, we revealed that high extracellular cystine concentration drives the antiproliferative effect of GAC0001E5 in PDAC cells. These findings support the idea of inhibiting LXR activity using inverse agonists and degraders for the treatment of advanced pancreatic cancer. This study also highlights a novel role for LXR in regulating glutamine metabolism and glutathione metabolism in pancreatic cancer. These novel LXR ligands can further be used to advance basic research on ligand design, allosteric mechanisms, and LXR functions in other cancer models.Item Characterization of the Pro-Survival Role of Kinase-Independent EGFR in Cancer(2017) Srivastava, Shivangi; Katreddy, Rajashekara; Thomas, Rintu; Zhang, WeihuaEGFR (Epidermal Growth Factor Receptor), a receptor tyrosine kinase is overexpressed or mutated in a variety of tumors of epithelial origin. Although, its kinase activity is well characterized and is being targeted in the clinic, still patients develop resistance and stop responding to TKIs. Therefore, there is a need to understand the kinase-independent functions of EGFR and how it can be clinically targeted. Here, I describe the work that tests the hypothesis that EGFR independent of its tyrosine kinase activity mediates survival of cancer cells under various physiological stresses and thereby promoting resistance against TKIs.Item Insights into the Substrate Binding Specificity of Quorum-Quenching Acylase PVDQ(2019-05) Liu, Yanyun 1987-; Briggs, James M.; Fox, George E.; Rosenzweig, Jason A.; Zhang, WeihuaThe N-acyl homoserine lactone acylase, PvdQ, from human opportunistic pathogen Pseudomonas aeruginosa is a quorum-quenching enzyme that can hydrolyze the amide bond of the quorum sensing signaling N-acyl homoserine lactones (AHLs) thereby degrading the signaling molecules, inhibiting the biofilm formation and reducing virulence gene expression. Previous studies demonstrated that PvdQ has different preferences for AHLs with different acyl chain lengths and substituents. However, the substrate binding specificity determinants of PvdQ with different bacterial ligands remain unknown and unintuitive. Elucidation of these determinants can lead to mutants with efficiency and broader substrate promiscuity. To investigate this question, a computational study was carried out combining multiple molecular docking methods, molecular dynamics (MD) simulations, residue interaction network analysis, and binding free energy calculations. The main findings are: firstly, results from pKa predictions support the observation that the pKa of the N-terminus of Serβ1 was depressed due to the surrounding residues. Multiple molecular docking studies provided information about PvdQ binding modes and binding affinities. Secondly, analysis of the protein dynamic fingerprint of each complex from MD simulations demonstrated that binding of C12-homoserine lactone (C12-HSL) ligand reduced the global motion of the complex and maintained the correct arrangement of the catalytic site. Further, the residue interaction network analysis of each system illustrated that there are more communication contacts and pathways between the residues in the C12-HSL complex as compared to other complexes. The binding of the C12-HSL ligand facilitates structural communication between the two knobs and the active site. The binding of other ligands tends to impair these specific communication pathways, leading to a catalytically inefficient state. Finally, simulation results from free energy landscape and binding free energy analysis revealed that the C12-HSL ligand has the most favorable binding free energy and greater stability than the less favored ligands. Each of the following residues: Serβ1, Hisβ23, Pheβ24, Metβ30, Pheβ32, Leuβ50, Asnβ57, Thrβ69, Valβ70, Trpβ162, Trpβ186, Asnβ269, Argβ297 and Leuα146, play different roles in substrate binding specificity. This is the first computational study that provides molecular information for structure-dynamic-function relationships of PvdQ with different bacterial ligands and demonstrates determinants of substrate binding specificity.Item Ligands of Estrogen Receptor β in Prostate and Brain(2014-05) Muthusamy, Selvaraj 1980-; Gustafsson, Jan-Åke; Moore, David; Warner, Margaret; Zhang, WeihuaEstrogen receptor β is a nuclear receptor expressed in various tissues in the body including prostate and brain. In addition to 17β-estradiol, other steroids like 5α-androstane-3β, 17β-diol (3β-Adiol) and 5-androstene-3β, 17β-diol have been reported to be endogenous ligands of ERβ. The concentration of these ligands in the tissue and the metabolism of these ligands by different enzymes regulate the transcriptional activity of ERβ. In human prostate, 3β-Adiol acts on ERβ to exert an anti-proliferative effect to counteract the proliferative activity of 5α-dihydrotestosterone (DHT) mediated through androgen receptor. 3β-Adiol is produced from 5α-dihydrotestosterone. In chapter 2, we show that 17β-HSD6, a predominant enzyme expressed in human prostate, converts DHT to 3β-Adiol. This conversion of DHT to 3β-Adiol is capable of activating ERβ at physiological concentrations of DHT. Immunohistochemical analysis revealed that 17β-HSD6 is expressed in ERβ-positive epithelial cells of the human prostate and that, both ERβ and 17β-HSD6 were present in benign prostatic hyperplasia (BPH) samples and were undetectable in prostate cancers of Gleason grade higher than 3. These observations reveal that formation of 3β-Adiol via 17β-HSD6 from DHT is an important growth regulatory pathway that is lost in prostate cancer. ERβ plays an important role in development and homeostasis of brain. In chapter 3, using liquid extraction, solid phase extraction and LC-MS/MS, we show that estrone is the only detectable endogenous ligand of ERβ in mouse brain tissue. We also show that CYP7B1 knockout mice have significantly higher level of estrone compared to the sex matched wild type controls. We show that only 25% of estrone is converted to estradiol by brain tissue after 24 h of incubation, which indicates that the conversion of estrone to estradiol by brain tissue is very slow. Using cell based transactivation assays we show that estrone is capable of activating ERβ with an EC50 of 3.5 nM. Since the concentration of estrone in mouse brain is 25 nM to 35 nM, estrone could be a physiological ligand of ERβ in mouse brain.Item Novel Mechanisms of Kinase Independent Pro-Survival Functions of EGFR in Cancer(2017-12) Katreddy, Rajasekhara Reddy 1989-; Zhang, Weihua; Bark, Steven J.; Chung, Sang-Hyuk; Gan, Boyi; Bhattacharya, Pratip K.Tyrosine kinase activity of epithelial growth factor receptor (EGFR) plays an important role in regulating numerous signaling pathways in cancer. It is commonly overexpressed in various cancers. Several strategies for cancer therapy were developed to target the kinase function of EGFR. However, the chemotherapeutic drugs against EGFR kinase functions developed either acquired resistance or innately resistant. Over past decade, few reports have demonstrated that EGFR possesses kinase-independent pro-survival functions. A better understanding of the molecular mechanisms involved in the kinase-independent pro-survival functions of EGFR bears a great potential for developing new strategies for cancer therapy. Therefore, this study was designed to further explore the molecular mechanisms underlying kinase-independent pro-survival functions of EGFR. Earlier, we have reported autophagy induced by EGFR knockdown (loss-of-EGFR) caused cell death whereas autophagy induced by EGFR’s kinase inhibition caused growth arrest. The differences in autophagy mechanisms were unknown. In the first approach, we have shown that loss-of-EGFR causes selective mitophagy mediated by EGFR’s kinase-independent interaction with RICTOR and UT2 and activation of mTORC2/Akt signaling whereas inhibition of EGFR kinase function causes non-selective macro autophagy mediated by activation of mTORC1 signaling. To further support this, we have found an EGFR downregulating peptide demonstrating the induction of mitophagy and inhibition of tumor growth of orthotopic ovarian cancers in mice. It was known that EGFR interacts with SGLT1 and stabilizes it in a kinase-independent manner to maintain the intracellular glucose concentration and inhibiting SGLT1 activity sensitized prostate cancer cells to EGFR TKIs. However, the role of SGLT1 in EGFR kinase inhibited conditions were not known. In the second approach of this dissertation, we have demonstrated that SGLT1 undergoes oligomerization and upon treatment with EGFR TKIs, enhances its oligomerization and upregulates sodium-dependent glucose transport. We also showed that increase in SGLT1 oligomerization was sustained in EGFR TKI resistant cells and inhibition of SGLT1 activity with phlorizin reduced the survival of resistant cells. It is concluded that enhanced SGLT1 activity mediated by oligomerization in EGFR TKI environment is another survival mechanism and may serve as a potential target for cancer therapy.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 Role of De Novo Fatty Acid Synthesis in Intrinsic Activation of EGFR in Cancer(2014-08) Bollu, Lakshmi Reddy 1983-; Zhang, Weihua; Khurana, Seema; Widger, William R.; Peng, HuangDe novo fatty acid synthesis is one of the major upregulated metabolic pathways in cancer. De novo fatty acid synthesis pathway is crucially involved in cell proliferation, survival and drug resistance of cancer cells. This study focused on the role of fatty acid synthesis in intrinsic activation of EGFR and drug resistance in cancer. EGFR is a receptor tyrosine kinase expressed in most human cancers of epithelial origin and advanced cancers. Besides the plasma membranous (pmEGFR) and nuclear localization, EGFR can also exist in mitochondria (mtEGFR). Our studies revealed that EGFR exists in the inner mitochondrial membrane of prostate and breast cancer cells and promotes mitochondrial fusion through increasing the protein levels of PHB2 and OPA1. Interestingly, in this study, we found that activated pmEGFR activates mtEGFR through de novo synthesized palmitate, through palmitoylation, and promotes mitochondrial fusion. Along with mtEGFR, we also found that pmEGFR also undergo de novo fatty acid synthesis dependent palmitoylation which is important for EGFR localization, stability and signaling. We also observed intrinsic activation of EGFR in response to anticancer drugs such as SN38 and oxaliplatin dependent on de novo fatty acid synthesis. Finally, targeting de novo fatty acid synthesis or palmitoylation significantly increased the sensitivity of cancer cells to EGFR TKIs or anticancer drugs. In conclusion, through this study we’ve uncovered the importance of de novo fatty acid synthesis in intrinsic activation and non-classical functions of EGFR in cancer.Item The Role of Kinase Inactive EGFR in Cancer Cell Survival(2019-05) Thomas, Rintu Thankam 1989-; Zhang, Weihua; Bawa-Khalfe, Tasneem; Chung, Sang-Hyuk; Lu, XiongbinInhibiting the tyrosine kinase activity of epidermal growth factor receptor (EGFR) using small molecule tyrosine kinase inhibitors (TKIs) are often ineffective in treating cancers harboring wild-type (wt) EGFR. Given the fact that EGFR possesses kinase independent pro-survival function, the role of TKI inactivated EGFR in cancer cell survival needs to be addressed. In this study, we chronically treated wt EGFR expressing cancer cells, A549 (lung), DU145 (prostate), PC3 (prostate), and MDA-MB231 (breast), with TKIs until they acquired resistance to treatment. The primary focus of this thesis was to then to 1) characterize both the kinase activity and TKI induced dimerization status of EGFR in TKI resistant cells, 2) elucidate the biochemical nature of TKI induced EGFR dimers, and 3) determine the dependence of TKI resistant cells on kinase inactivated EGFR for survival. A substantial inhibition of EGFR’s phosphorylation/activation status coupled with an increase in TKI induced membrane tethered EGFR dimerization were observed in the TKI resistant cells. We demonstrated that TKI-induced EGFR dimerization is dependent on palmitoylation, independent of EGFR’s kinase activity, and mutations of cysteine residues known to be critical for EGFR’s palmitoylation abolished TKI-induced EGFR dimerization. Disruption of TKI induced dimerization by inhibition of palmitoylation, or targeted reduction of the kinase inactivated EGFR by siRNA or by an EGFR downregulating peptide were observed to be lethal to TKI resistant cancer cells. Taken together, the data presented in this thesis suggests that the kinase-inactivated EGFR remains to be a viable therapeutic target for wt EGFR cancers and that inhibiting palmitoylation or downregulating EGFR may overcome TKI resistance.Item Understanding Structure and Dynamics of PTEN and its Possible Genotype-Phenotype Correlations in Endometriosis and Cancer(2016-12) Smith, Iris Nira 1978-; Briggs, James M.; Zhang, Weihua; Heard, Michael J.; Gao, XiaolianThe phosphatase and tensin homolog deleted on chromosome 10, (PTEN) gene encodes a tumor suppressor phosphatase frequently mutated in various human cancers. Somatic missense mutations of PTEN have recently been found in patients with endometriosis, endometrial cancer, and ovarian cancer. Here we present the first computational analysis of 13 somatic missense PTEN mutations to assess a possible genotype-phenotype correlation in endometriosis and cancer. We posit PTEN’s active site defines a possible mutation-driven allosteric region wherein a subset of mutations correlate with endometriosis, endometrial cancer, and ovarian cancer. Our data suggest that mutations within the active site disrupt the structural stability, electrostatic interaction, global dynamics and the structural communication pathway, likely contributing to the aforementioned phenotypes. Multiple in silico prediction methods were utilized to calculate protein structural stability changes produced by each mutation; decreases in protein structure stability were seen in each mutation with an increase in dynamics across the phosphatase-C2 domain interface of R130G/L/Q and R173C/H mutations. To assess the impact on intrinsic and global dynamics, elastic network models (ENMs) were employed demonstrating changes from wild-type “hinge-bending” to “zipper-like” global motions induced by each mutation. All-atom molecular dynamics (MD) simulations revealed large conformational changes that affect the global dynamics of the active site loops and the CBR3 loop in the C2 domain. Interestingly, mutations G36E/R, C124S, G129R, R130L/Q, R173C/H, and V191A dramatically affected the principal motions of the active site loops and inter-domain interface. Overall, the global dynamics induced by each mutation effects reveal unique long-range perturbations that may impair PTEN’s function. We further investigated structural communication within each mutant system using protein structure network (PSN) analysis and found that R130 and R173 play critical roles in controlling salient communication pathways suggesting a compelling interplay between the two positions involving a potential mutation-driven allosteric interface. The results of this research provide a greater understanding of the mechanistic role of mutated PTEN associated with endometriosis and cancer. It is our hope that these results will aid in a better clinical-molecular classification of the resulting phenotypes allowing for translation into improved diagnostic and therapeutic approaches.Item Unraveling the Underlying Mechanism of Bcl-2 Mutations Regulated Apoptotic Blocks in Diffuse Large B-Cell Lymphoma (Dlbcl) Patients(2017-12) Singh, Khushboo 1987-; Briggs, James M.; Fox, Robert O.; Zhang, Weihua; Willson, Richard C.The BCL-2 proteins have been intensively investigated for the past few decades owing to its importance in regulating the intrinsic pathway of apoptosis. Deregulation of apoptosis is implicated in the pathogenesis of cancer and in the sensitivity of tumors to therapeutic interventions. Genome-sequencing data obtained from a large series of DLBCLs have identified frequent mutations in the translocated BCL2 gene. However, the heterogeneity of the mutation profile complicates the prediction of the functional effects of the DLBCL-specific mutations. The aim of the research is to present an integrated snapshot of recurrent missense mutations in the BCL2 gene reported in independent genomic studies and to explore the various ways these mutations could affect the binding of proteins and inhibitors leading to the activation of oncogenic pathways and drug resistance. Recognition of these pathways could have crucial ramifications for our ability to understand inter-patient heterogeneity of BCL2 mutations, thereby, providing avenues for tailored treatment of DLBCL. A considerable amount of evidence suggests the preferential clustering of mutations in the BH4 domain of the BCL-2 protein. The biochemical and structural properties of the BH4 domain enables the binding of the BH3 domain in the hydrophobic groove of the protein without being a part of the groove by stabilizing the core of the protein, thereby, maintaining the integrity of the groove. The incidence of N11Y mutation present in the BH4 domain has been reported in more than three independent studies, indicating its probable role in DLBCL pathogenesis. The impact of the N11Y mutation on the binding of a ‘BH3 mimetic’ was elucidated by molecular dynamics studies. Mutation of N11Y results in an alteration of the shape of the hydrophobic groove, subsequently changing the ligand orientation and counteracting the phenomenon of unwinding of the LB region, a crucial event observed in the wild type model. Principal component analysis captured a stretching motion of the hydrophobic groove for efficient ligand accommodation in the wild-type complex but not in the mutant model. Overall, this study unravels a probable mechanism of drug resistance induced by a BCL-2 mutation, which could be of great relevance while designing and tailoring therapeutics.