• A Molecular Basis of Chemoresistance in Bladder Cancer

      Lahorewala, Sarrah; Biochemistry and Cancer Biology (Augusta University, 2020-12)
      Background: In advanced bladder cancer (BC), development of resistance to the frontline chemotherapeutic drugs Gemcitabine and Cisplatin contributes to the poor prognosis of patients. Newly discovered chondroitinase, HYAL-4 V1 (V1), drives malignant transformation in BC. We evaluated V1’s role and the downstream molecules involved in the mechanistic regulation of chemoresistance in BC. Experimental Design: HYAL-4 expression was evaluated by RT-qPCR and IHC in metastatic muscle-invasive BC patients who received Gemcitabine plus Cisplatin chemotherapy. HYAL-4 wild-type and V1 were stably expressed or silenced in three BC and one normal urothelial cell line. Transfectants were analyzed for Gemcitabine and Cisplatin sensitivity, and for Gemcitabine influx and efflux to determine the mechanism of Gemcitabine resistance. The effect of cytidine deaminase (CDA) inhibition on Gemcitabine sensitivity was evaluated in vitro and in xenograft models. Results: HYAL-4 expression was an independent predictor of disease-specific mortality and treatment failure in our clinical cohort, and stratified patients into higher risk for both those outcomes. V1-expressing BC and normal urothelial cells were resistant to Gemcitabine due to the upregulation of cytidine deaminase (CDA) expression and activity, resulting in increased Gemcitabine metabolism and efflux; treating cells with tetrahydrouridine (THU), a CDA inhibitor, abrogated the chemotherapeutic resistance. Gemcitabine-resistant V1 cells demonstrated increased expression of V1’s substrate CD44 and phosphorylated STAT3. Si-RNA mediated CD44 knockdown and STAT3 inhibition both sensitized cells to Gemcitabine in vitro. In xenograft models, treatment with a combination of Gemcitabine and THU completely inhibited tumor growth. Conclusions: This project discovered V1 as a novel determinant of Gemcitabine resistance and potential predictor of treatment response in BC. V1 drives resistance to Gemcitabine through CD44-STAT3 mediated upregulation of CDA, and inhibiting this pathway sensitizes tumor cells to the therapy in preclinical models of BC.
    • The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

      Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
      Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.

      Ibrahim, Mohammed Mahmoud Labib; Biomedical Sciences (Augusta University, 2019-05)
      A causal link between chronic inflammation and tumorigenesis is now well established in the literature with a great deal of supporting evidences from genetic, epigenetic, pharmacological and epidemiological perspectives. In particular, inflammatory bowel diseases represent an important risk factor for colon cancer development. Moreover, it seems that even sporadic colon cancers that do not develop as a complication of chronic colitis are also driven by inflammation. However, the molecular mechanisms behind inflammation-mediated colon tumorigenesis have remained largely unknown. Colitis associated cancer development is thought to be multifaceted due to a combination of genetic and epigenetic aberrations. Recently, epigenetic alterations -particularly aberrant DNA methylation- have gained great attention in cancer biology and have been observed to play a key role in the pathogenesis of inflammation-associated tumors; especially in colitis-associated cancer. IRF8, a key transcription factor originally identified in myeloid cells, has been reported to play a crucial role in myeloid cells differentiation and immune response regulation. IRF8 deficiency is associated with deregulation of myeloid cell differentiation and accumulation of immature myeloid subsets phenotypically and functionally resemble MDSCs (Myeloid Derived Suppressor Cells). IRF8 is thought to function as a tumor suppressor and was found to be silenced in different types of cancers including colon cancer. Myeloid derived-IRF8 has been extensively studied. However, the role of epithelial-derived IRF8 in colon inflammation and colon cancer initiation remains a point to be addressed. In this study, we generated conditional Irf8cKO mice in which IRF8 is specifically deleted in colon epithelium. Irf8cKO mice exhibit a more aggressive pattern of colitis associated cancer with higher tumor incidence and severe loss of body weight. Additionally, we provide evidence that chronic inflammation promotes the accumulation and infiltration of CD11b+Gr1+ MDSCs, which plentifully secrete IL10 in colon tissue. IL10 then induces STAT3 phosphorylation and nuclear translocation to bind to Dnmt1 and Dnmt3b promoters to upregulate their expression, leading to DNA hyper-methylation at the Irf8 promoter to silence IRF8 expression in colonic epithelial cells and promote colon tumorigenesis. Collectively, our data pinpoint the MDSC-IL10-STAT3-DNMT3b-IRF8 axis as a novel bridge between chronic inflammation and colon cancer formation.
    • Gene regulation by the putative Campylobacter jejuni diguanylate cyclase CbrR

      Fulmer, Claudia; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
      As a leading cause of bacterial gastroenteritis, Campylobacter jejuni incurs health care costs estimated at $290 million a year in the US and up to 40,000 deaths in children aged 5 and younger worldwide. As such, determining those proteins that regulate C. jejuni virulence factors are prime targets to possibly develop a prophylactic therapy, that as of yet does not exist. CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), the class of enzyme that catalyzes the synthesis of cyclic di-GMP, a universal bacterial second message molecule, from GTP. In C. jejuni strain DRH212, an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+, were constructed. Soft agar motility tests, biofilm formation assays, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed. Site-directed mutagenesis was performed on cbrR to make alanine substitutions in both the autoinhibitory site (I-site) and active site and differential radial capillary of action ligand (DRaCALA) assays were performed to determine nucleotide binding by wild-type CbrR and the CbrR point mutants. Soft agar motility assays indicated a hyper-motile phenotype associated with the C. jejuni cbrR- mutant, whereas motility was all but negated in the cbrR+ complement. Biofilm assays and SEM demonstrated similar formation and robustness of biofilms between wild type and cbrR- mutant, however cbrR+ was unable to form significant biofilms in 72 hours. TEM images showed similar cellular morphology between cbrR-, wild type, and cbrR+, however cbrR+ cells had fewer flagella. DRaCALA assays showed wild-type CbrR and the active site mutant were both able to bind GTP and cyclic di-GMP, whereas the I-site mutant lost the ability to bind cyclic di-GMP, indicating the product binding site on CbrR. The highly conserved diguanylate cyclase CbrR is the only annotated DGC in the C. jejuni genome. Though the active site sequence is highly variant when compared to the consensus sequence, this protein is able to bind both substrate and product of the chemical synthesis of cyclic di-GMP and has now been shown to be a negative regulator of motility, a critical virulence factor in C. jejuni pathogenesis.

      Morera, Daley S; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
      There is a need for novel prognostic biomarkers and targeted treatments in bladder cancer (BC), even more so for muscle-invasive disease (MIBC). Discovery of molecular markers to predict outcome in BC patients may lead to identification of impactful therapeutic targets. The hyaluronic acid (HA) family of molecules and distinct molecular subtypes have both been investigated as potential prognostic markers. HA family and chondroitin sulfate proteoglycans, such as CD44, have been implicated in driving aggressiveness of disease; however, a chondroitinase enzyme that cleaves chondroitin sulfate proteoglycans has not been identified in any eukaryotic system. We evaluated molecular markers of BC and the first known eukaryotic/human chondroitinase, that we identified, for their ability to predict clinical outcome in patients, and for their roles as drivers of disease. We also investigated the anti-tumor effects of HA synthesis inhibitor 4-methylumbelliferone (4MU), a non-toxic orally bioavailable supplement. This study demonstrates that transcript levels of HA family members can predict metastasis and poor survival in BC patients. HA- family expression also correlated with epithelial mesenchymal transition (EMT) markers β -Catenin, Twist, Snail, and E-Cadherin. HA signaled through its receptors CD44/RHAMM and the PI3-K/AKT axis. 4MU targeted HA signaling, inhibiting proliferation and motility/invasion, inducing apoptosis in vitro, and preventing tumor growth in vivo. We discovered that a previously unidentified splice variant of HYAL-4 was elevated in bladder tumors. We named this variant "V1". Our studies showed that V1 had chondroitinase activity, cleaved chondroitin-6-sulfate from CD44, and consequently increased CD44 secretion. In vivo, V1-expressing urothelial cells formed muscle-invasive tumors and V1-expressing cancer cells developed metastatic tumors. Evaluation of the prognostic significance of the molecular subtypes of MIBC that were recently identified by other groups, showed the subtypes to have little to no predictive ability for clinical outcome in multivariate analyses that included standard clinical parameters. Consistently, clinical parameters such as histopathologic tumor grade, T-stage, and lymph node status, outperformed the molecular subtypes. Contrarily, V1 levels could independently predict metastasis and survival with high efficacy, suggesting that focusing on V1 as a functional biomarker may be a better strategy to improve clinical outcome of BC patients.
    • Study of Transport Systems for Oligopeptides

      Chothe, Paresh P.; Department of Biochemistry and Molecular Biology (2010-12)

      Abdelrahman, Ammar; Department of Biochemistry and Molecular Biology (Augusta University, 2020-12)
      Currently, treatments of diabetic retinopathy (DR) have limited therapeutic benefits and limited accessibility to the growing diabetic population at risk because of the high expenses and complicated procedures. Inflammation, endothelial dysfunction, and microvascular damage are common features of diabetic complications including DR. GPR109A is the metabolite sensing receptor of beta-hydroxybutyrate (BHB) the principal ketone body in humans. Our previous studies have shown the role of GPR109A expression in promoting anti-inflammatory response in retinal pigmented epithelial (RPE) cells and the relevance of the receptor in DR. Expression of the GPR109A in microvascular endothelial cells (ECs) has been reported recently. However, the relevance of GPR109A expression and activation to retinal EC functions are yet to be studied. Our goal in this study was to identify the role of GPR109A expression and activation in barrier and angiogenic functions of retinal ECs in context of diabetic retinopathy. We used electrical cell impedance sensing (ECIS) technology to evaluate barrier functions in primary human retinal endothelial cells (HRECs) which constitute the inner BRB. Knocking down GPR109A in HRECs with siRNA decreased the transendothelial electrical resistance (TEER) compared to scrambled siRNA. Treating HRECs with BHB increased their TEER and counteracted VEGF-induced barrier disruption through activation of GPR109A and increasing zonula occludens-1 (ZO-1) expression. Treatment of STZ-diabetic mice with exogenous BHB for one month protected against the pathologic albumin leakage induced by diabetes and improved the visual acuity of this animal model of diabetes. Using the mouse model of oxygen induced retinopathy (OIR), we showed that Gpr109a-/- mice had slower vascular recovery from pathologic angiogenesis compared to age matched wild type mice. Moreover, physiologic revascularization of vaso-oblitrated retinas was impaired by loss of GPR109a and associated with dysregulated inflammatory and angiogenic signaling. Collectively, these data point to a role for GPR109A in the regulation of barrier and angiogenic mechanisms in retinal ECs and, promote the receptor as a potential druggable target for impacting these mechanisms in microvascular retinal diseases such as DR.