Identifying Novel Inhibitors of Epithelial to Mesenchymal Transition (EMT) for Targeting Pancreatic Cancer Metastasis and Cancer Stem Cells

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, and it is expected to become the second-leading cause of cancer-related death by 2030. Currently, there are no early detection tests and most patients with localized disease have no recognizable symptoms. As a result, more than half of the patients with this disease are diagnosed at a stage where metastases have developed, for whom the overall 5-year survival is only 2%. Moreover, these tumors are highly enriched with a cancer stem cell (CSC) population (~1%), which is highly resistant to chemotherapeutic drugs, and therefore escapes chemotherapy and promotes tumor recurrence. Recent evidence suggests that epithelial to mesenchymal transition (EMT) is associated with metastasis, generation of CSCs, and treatment resistance in solid tumors including pancreatic cancer. Therefore, compounds inhibiting EMT hold the potential to reverse drug-resistance or inhibit metastasis and CSCs, and therefore could provide better treatment outcome for patients with pancreatic cancer. The overall goal of this dissertation is to investigate novel EMT inhibitors for targeting pancreatic cancer metastasis and CSCs. First we demonstrated in preclinical models that treatment with pharmacological doses of ascorbate resulted in inhibition of EMT, metastasis and CSCs. In addition, ascorbate decreased the expression of HDAC6, and inhibited activity of Sirt-2 by depleting NAD+ levels resulting in robustly increased α-tubulin acetylation in pancreatic cancer cells. Ascorbate mediated tubulin acetylation promoted α-tubulin polymerization and stabilization, mimicking the cellular outcomes of paclitaxel to inhibit cancer cell metastasis. Next, we investigated novel derivatives of the histone deacetylase (HDAC) inhibitors SAHA and MS-275, which are known EMT inhibitors. In an effort to increase efficacy and reduce toxicities of the HDAC inhibitors, we found that the novel synthetic compounds St-1 and St-3 potently inhibited pancreatic cancer cell proliferation and CSCs. St-1 has exhibited similar potency in HDAC inhibition compared to the parent compounds (SAHA and MS-275). Surprisingly, St-3 acted via totally different mechanisms from SAHA and MS-275. St-3 exhibited anti-tumor effects by blocking the interaction of human antigen R (HuR) with its target mRNAs. Finally, we established and performed a high throughput screening approach to identify inhibitiors of cancer cell EMT. 1-(benzylsulfonyl) indoline (BSI) was found to be a novel EMT inhibitor. BSI significantly inhibited pancreatic cancer cell migration invasion and CSCs. To enhance the efficacy of BSI, several analogues of BSI were tested for their activities on EMT and CSC inhibition. However, BSI analogues failed to show superior anti-migration or anti-CSC activities compare to BSI. In conclusion, this dissertation resulted in the identification of several novel EMT inhibitors, which can be tested further in preclinical studies for their anti-tumor efficacy

A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity

Searching for Binding Partners for the Novel PHKG1 Variant, PhKγ 181

Phopshorylase kinase (PhK) is a hexadecameric holoenzyme made up of four different subunits in the arrangement (αβγδ)4 and has total molecular mass of 1.3MDa. Alpha and β are regulatory subunits, γ is catalytic, and δ is an intrinsic molecule of calmodulin. PhK is a serine threonine kinase with glycogenolytic regulatory functions. Our lab has recently discovered that the γ subunit can be alternatively processed to produce a truncated form of 181 residues (γ-181). This variant of γ contains a phosphorylation site for PK-C, and its activity is influenced by this phosphorylation. We are using a LexA based yeast two hybrid system to identify potential binding partners for γ-181. In the present study we are cloning the human γ-181 in to a Lex-A DNA binding domain vector to use as “bait” for screening a human brain c-DNA library. Interaction between γ-181 and the brain c-DNA library proteins will be monitored using LacZ and LEU2 as reporter genes. Library plasmids isolated from the clones that show positive results on the yeast two hybrid screen will be sequenced to identify novel binding partners for γ-181

Metabolomic Profiling of Red Blood Cells to Identify Molecular Markers of Methotrexate Response in the Collagen Induced Arthritis Mouse Model

Although methotrexate (MTX) is the first line disease-modifying therapy used in the treatment of autoimmune arthritis, it is limited by its unpredictable and variable response profile and lack of therapeutic biomarkers to predict or monitor therapeutic response. The purpose of this work is to evaluate the utility of red blood cell (RBC) metabolite profiles to screen for molecular biomarkers associated with MTX response. Methods: Utilizing the collagen-induced arthritis mouse model, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and RBC samples were collected and analyzed by semi-targeted global metabolomic profiling and analyzed by univariate analysis. Results: MTX treatment normalized the following RBC metabolite levels that were found to be altered by disease induction: N-methylisoleucine, nudifloramide, phenylacetylglycine, 1-methyl-L-histidine, PC 42:1, PE 36:4e, PC 42:3, PE 36:4e (16:0e/20:4), and SM d34:0. Changes in the RBC metabolome weakly but significantly correlated with changes in the plasma metabolome following MTX treatment (ρ = 0.24, p = 1.1 × 10−13). The RBC metabolome resulted in the detection of nine significant discriminatory biomarkers, whereas the plasma metabolome resulted in two. Overall, the RBC metabolome yielded more highly sensitive and specific biomarkers of MTX response compared to the plasma metabolome. N-methylisoleucine was found to be highly discriminatory in both plasma and RBCs. Conclusions: Our results suggest that RBCs represent a promising biological matrix for metabolomics and future studies should consider the RBC metabolome in their biomarker discovery strategy

Metabolomic Profiling of Red Blood Cells to Identify Molecular Markers of Methotrexate Response in the Collagen Induced Arthritis Mouse Model

Although methotrexate (MTX) is the first line disease-modifying therapy used in the treatment of autoimmune arthritis, it is limited by its unpredictable and variable response profile and lack of therapeutic biomarkers to predict or monitor therapeutic response. The purpose of this work is to evaluate the utility of red blood cell (RBC) metabolite profiles to screen for molecular biomarkers associated with MTX response. Methods: Utilizing the collagen-induced arthritis mouse model, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and RBC samples were collected and analyzed by semi-targeted global metabolomic profiling and analyzed by univariate analysis. Results: MTX treatment normalized the following RBC metabolite levels that were found to be altered by disease induction: N-methylisoleucine, nudifloramide, phenylacetylglycine, 1-methyl-L-histidine, PC 42:1, PE 36:4e, PC 42:3, PE 36:4e (16:0e/20:4), and SM d34:0. Changes in the RBC metabolome weakly but significantly correlated with changes in the plasma metabolome following MTX treatment (ρ = 0.24, p = 1.1 × 10−13). The RBC metabolome resulted in the detection of nine significant discriminatory biomarkers, whereas the plasma metabolome resulted in two. Overall, the RBC metabolome yielded more highly sensitive and specific biomarkers of MTX response compared to the plasma metabolome. N-methylisoleucine was found to be highly discriminatory in both plasma and RBCs. Conclusions: Our results suggest that RBCs represent a promising biological matrix for metabolomics and future studies should consider the RBC metabolome in their biomarker discovery strategy

Methotrexate Disposition, Anti-Folate Activity, and Metabolomic Profiling to Identify Molecular Markers of Disease Activity and Drug Response in the Collagen-Induced Arthritis Mouse Model

Methotrexate (MTX) is widely used in the treatment of autoimmune arthritis but is limited by its unpredictable and variable response profile. Currently, no biomarkers exist to predict or monitor early therapeutic responses to MTX. Using a collagen-induced arthritis (CIA) mouse model, this study aimed to identify biochemical pathways and biomarkers associated with MTX efficacy in autoimmune arthritis. Following arthritis disease induction, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and disease activity was assessed based on disease activity scores (DAS) and paw volume (PV) measurements. Red blood cell (RBC) and plasma samples were collected at the end of the study and were assessed for folate and MTX content. Plasma samples were analyzed by semitargeted global metabolomic profiling and analyzed by univariate and multivariate analysis. Treatment with MTX was associated with significant reductions in disease activity based on both DAS (p = 0.0006) and PV (p = 0.0006). MTX therapy resulted in significant reductions in 5-methyltetrahydrofolate (5mTHF) levels in plasma (p = 0.02) and RBCs (p = 0.001). Reductions in both RBC and plasma 5mTHF were associated with lower DAS (p = 0.0007, p = 0.01, respectively) and PV (p = 0.001, p = 0.005, respectively). Increases in RBC MTX were associated with lower DAS (p = 0.003) but not PV (p = 0.23). Metabolomic analysis identified N-methylisoleucine (NMI) and quinolone as metabolites significantly altered in disease mice, which were corrected towards healthy control levels in mice treated with MTX. Reductions in plasma NMI were associated with lower DAS (p = 0.0002) and PV (p = 9.5 × 10−6). Increases in plasma quinolone were associated with lower DAS (p = 0.02) and PV (p = 0.01). Receiver-operating characteristic curve analysis identified plasma NMI (AUC = 1.00, p = 2.4 × 10−8), RBC 5mTHF (AUC = 0.99, p = 2.4 × 10−5), and plasma quinolone (AUC = 0.89, p = 0.01) as top discriminating metabolites of MTX treatment. Our data support a relationship between MTX efficacy and its effect on circulating folates and identified 5mTHF, NMI, and quinolone as potential therapeutic biomarkers of disease activity and MTX response in the CIA mouse model of autoimmune arthritis

Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation.

Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition.An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay.Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities.This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as anti-EMT and anti-CSC drug leads