Qiao-Hong Chen, CSU Fresno - Chemistry/Biochemistry
An amide mimic of desTHPdactylolide: Total synthesis and antiproliferative evaluation
Dactylolide serves as an intriguing synthetic target because of its structural resemblance to zampanolide, a marine microtubule stabilizing macrolide with a very promising anticancer potency and a unique covalent-binding mechanism. In our previous investigation, desTHPdactylolide has been synthesized in both (17S) and (17R) configurations. And (17R) configuration was identified as the eutomer in suppressing prostate cancer cell proliferation. The current study aims to synthesize an amide mimic of desTHPdactylolide that could be metabolically more stable in comparison with desTHPdactylolide. To this end, the amide mimic of (17R) desTHPdactylolide has been successfully synthesized through a 26-step transformation, using commercially available 2-butyn-1-ol as starting material. Our WST-1 cell proliferation assay in three human prostate cancer cell models indicated that the stable lactam can serve as the bioisostere for the lactone in desTHPdactylolide. This presentation will report the full details of the synthesis and antiproliferative activities of the amide mimic.
Lucas Luna (Graduate), San Diego State - Chemistry/Biochemistry
Kinetic and Cellular Consequences of pH on IDH1 Activity
Changes in the intracellular environment, such as changes in pH, oxygen levels, oxidative stress can reroute metabolism by altering the activities of metabolic enzymes like isocitrate dehydrogenase 1 (IDH1). IDH1 is responsible for catalyzing the reversible NADP+-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and NADPH. Amplification of IDH1 is associated with cancer, including 65% of primary glioblastomas. Protein activity in health and disease can be regulated by the cellular environment to respond to the needs of the cell often through post-translational modifications (PTMs). Protein protonation, an underappreciated PTM, can have drastic effects on enzyme activity and metabolites present. We kinetically assessed the effect of pH on recombinant WT IDH1 by monitoring the increase or decrease in absorbance of NADPH at 340 nm using a spectrophotometer. We found that the rate of the forward reaction was pH-dependent and decreases as the pH becomes more acidic. However, the rate of the reverse reaction does not show pH dependence. In cellular assays, we treated HT1080 cells with proton pump inhibitors to lower intracellular pH. Thus far, esomeprazole has the best effect, lowering the intracellular pH from 7.57 to 7.28. Overall, the data elucidates the effect of pH on IDH1 catalytic activity and the cellular consequences that pH has metabolites present.
Jacqueline Lara (Graduate), San Diego State - Biology
The role of the NF-kB pathway in ovarian cancer tumor-initiating cells (TICs)
Ovarian cancer is the most lethal gynecological malignancy in the United States. Most patients initially respond to platinum-based chemotherapy; however, over 70% of advanced stage tumors relapse. Recent studies suggest a minority population of primitive cells, termed tumor-initiating cells (TICs), can resist chemotherapy and re-create heterogeneous tumors to cause relapse. Identifying the mechanisms that support this elusive population will be critical for preventing relapse. Our lab is focused on clarifying the role of NF-kB transcription factors, RelA and RelB, in ovarian TICs. NF-kB is a ubiquitous signaling complex aberrantly activated in over half of ovarian cancers. Our preliminary data suggest RelA supports cellular activities such as differentiation and proliferation, whereas RelB supports quiescence and drug resistance. We have performed RNA- and ChIP-sequencing analysis of RelA and RelB targets to define the gene sets regulated by these transcription factors in ovarian cancer cells. We present here our recent data including flow cytometry, spheroid formation, proliferation assays, and chemoresistance studies that further clarify how these transcription factors regulate diverse gene sets to maintain ovarian TICs. A better understanding of NF-kB signaling in TICs will guide the design for more effective therapies to overcome chemoresistance and relapse, and improve survival of women with ovarian cancer.
Cameron Geller (Graduate), CSU, Northridge - Biology
eIF5A1/2-focused Bioinformatic Analyses Suggest a Role for ECM-mediated Nucleocytoplasmic Transport in Translational Control of Non-Canonical TGFb Signaling
Eukaryotic initiation translation factor 5A (eIF5A1/2) undergoes a unique, post-translational hypusine modification involving deoxyhypusine synthase (DHPS) followed by exportin 4 (XPO4)-mediated nucleocytoplasmic transport. However, mechanisms governing this transport are unclear. Our work highlights DHPS-dependent eIF5A1/2 hypusination inhibition blocks Pseudopodium-Enriched Atypical Kinase One (PEAK1)-dependent non-canonical transforming growth beta (TGFb)/fibronectin-mediated breast cancer metastasis. Additionally, our most recent experiments reported fibronectin stimulates nucleocytoplasmic transport of eIF5A1/2 in a DHPS-dependent manner. To identify new transport mechanisms, hypusination pathway and cell heterogeneity markers were input into Cytoscape Agilent Literature Search, generating an interactome containing eIF5A1/2-interacting genes. Analyzing Copy Number Analysis and RNA-seq data from over 2000 breast cancer patients on Cancer Bioportal revealed decreased survival in patients with SMAD3 amplification and high SLC3A2 expression. Interestingly, SLC3A2 is known to potentiate fibronectin-integrin signaling and cell survival, while SMAD3 is a canonical TGFb signaling pathway transcription factor whose activity is dampened by XPO4. Together, this work reveals a novel ECM-mediated transport of eIF5A1/2 that promotes PEAK1 translation and non-canonical TGFb signaling. Targeting this mechanism holds promise for abrogating disease phenotypes including cancer metastasis.
Jonas Onuoha (Graduate), CSU, Fresno - Biology
Developing and optimizing mixed polymeric micelles to target chemoresistant pancreatic cancer
Pancreatic ductal adenocarcinoma has the highest mortality rates among all cancer types. This has been attributed to dismal prognosis, largely linked to aberrant chemo-resistance and complicated molecular mechanisms associated with disease progression. Many studies have demonstrated the need for improved strategies to specifically target tumor cells by delivering a higher concentration of anticancer agent either as a single dose or in combination with gold standard chemotherapy to maximize the synergistic effect and improve treatment outcomes. Mixed polymeric micelle drug delivery systems have been shown to increase the solubility and bioavailability of poorly water-soluble anticancer agents such as the plant polyphenol, curcumin. We prepared and tested curcumin-loaded mixed polymeric micelle formulations of poloxamer 407/TPGS and evaluated their physiochemical properties. Optimized formulations had acceptable particle sizes, negatively charged zeta potential, good curcumin loading capacity and high encapsulation efficiency. Preliminary in vitro cytotoxicity assays show robust cell killing in AsPC-1 cells. Overall, the improved formulation will potentiate the chemotherapeutic effect of curcumin and other anticancer agents like gemcitabine in treating drug-resistant pancreatic cancer cells. Future studies will determine cellular uptake and delineate the molecular mechanism for increased sensitivity of the drug-resistant pancreatic cells to the combinatorial therapy.
Jonathan Chacon (Graduate), CSU, Los Angeles - Biology
Concurrent activation of AMPK and mTOR signaling
AMPK and mTOR signaling pathways are canonically mutually antagonistic, with AMPK promoting energy conserving processes and mTOR promoting energy expensive processes. However, in some contexts, AMPK and mTOR signaling are found to be concurrently activated, although the biological significance of these contexts is not well-described. This project will utilize transcriptomic and metabolic workflows to evaluate changes in gene regulation (using RNA-sequencing data) and the downstream consequences of these differential gene expression changes (using GC-MS data) to broadly document and contextualize concurrent activation of AMPK and mTOR signaling pathways. Understanding the various paths that cells may utilize to circumvent, override, or modify mechanisms promoting AMPK-mTOR mutual antagonism may provide a deeper understanding as to how concurrent activation of AMPK and mTOR signaling status is achieved. This, in itself, lends numerous opportunities to explore diseases which feature concurrent activation of AMPK and mTOR signaling from paradigms which up until now have remained elusive. Additionally, the identification and organization of unique parallels between various approaches that cells take to achieve concurrent activation of AMPK and mTOR signaling status may also open up new therapeutic avenues for disorders particularly cancer, in which AMPK and mTOR activation have both been shown to promote oncogenic processes.
Farhana Runa (Post-doc), CSU, Northridge - Biology
Role of RAI14 in collagen processing for the progression of pancreatic cancer
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors with a long-term survival rate of ~8%. A major limitation for the treatment of PDAC depends on the lack of understanding of the mechanisms of PDAC progression and metastasis as well as actionable targets for the drugs. We previously identified that RAI14 (Retinoic Induced Acid fourteen, Ankycorbin or NORPEG), a pseudopodium-enriched protein which is co-expressed with collagen and integrin a1. Further, we report that RAI14 associated with the actin cytoskeleton and promotes Src kinase stability, proliferation, and cell migration. Although a recent finding demonstrating that RAI14 and other n-ank proteins bind domains of high curvature and shape cell membrane, the actual function of RAI14 in PDAC development and progression is unknown. Here, using CancerBioPortal database, we identified RAI14 co-expressors across malignancies of digestive and reproductive systems. Further, we identified predicted protein-protein binding partners of RAI14 and RAI14-co-expressors using the ScanSite database. Analysis of the Cytoscape interactome and DAVID gene ontologies suggest that RAI14 may have potential functions as a regulator of collagen trafficking/deposition. The role of RAI14 is further examined on ECM of PDAC cell derivatives developed by CRISPR/Cas9 deletion methods. Our current and prospective findings have the potential to illuminate new mechanisms on PDAC progression.
Alina Simonyan (Undergraduate), CSU, Northridge - Biology
Development of an assay to detect matrix metalloproteinase inhibitors expressed by tumor-targeted Salmonella
Matrix metalloproteinases are proteolytic enzymes that are often over-expressed in tumors and contribute to metastasis through degradation of extracellular collagen matrix. Matrix metalloproteinase overexpression correlates with poor prognosis in a number of tumor types, and has been investigated as a therapeutic target. However, clinically, MMP inhibitors have shown toxicities towards organs such as the kidneys, which could prevent its use clinically. One way to improve the off-target effects of MMP inhibitors would be to limit their delivery to non-target sites. Tumor-targeted bacteria such as Salmonella VNP20009 selectively target tumors at ratios exceeding 1000:1 and offer the potential to locally produce and deliver MMP inhibitors within the tumors without off-target effects. However, in order to detect inhibition of proteolytic activity by VNP20009, development of an assay compatible with whole, live bacteria is desirable. We cloned the full-length human MMP-2 into autolytic strains of E. coli as potential sources of enzyme. We are investigating the use of co-culture of our MMP-2 producing lytic strains with our tumor-targeted Salmonella expressing MMP inhibitor peptides expressed as fusions with the carrier protein YebF in order to demonstrate MMP inhibition. We are also exploring further stimulation of MMP-2 proteinase production through a mercuric activation procedure.
Muthu Malavika Sugumar (Undergraduate), CSU, Northridge - Biology
Tryptophanase Expressed by Tumor-Targeted Salmonella Halts Breast Cancer Cell Growth In Vitro
Tryptophan is an essential amino acid required for growth and is also the precursor to kynurenine, an immunosuppressive molecule that plays a role in limiting anticancer immunity. Tryptophanase (TNase) is an enzyme expressed by different bacterial species that converts tryptophan into indole, pyruvate and ammonia, but is absent in the tumor-targeted Salmonella VNP20009. We cloned the E. coli TNase operon tnaCAB into the VNP20009 and were able to detect measurable indole using Kovács reagent. In order to conduct further experiments using the whole bacteria, we added the bacteriostatic antibiotic gentamycin to stop bacterial replication. Using 1 x10e8 CFU/mL bacteria as a fixed number, we found that there was no significant effect of gentamycin on the VNP20009- tnaCAB production of indole over time. Using limiting tryptophan, 1 x10e8 CFU/mL bacteria were able to convert as little as 1 μM tryptophan into measurable indol, and the bacteria were able to deplete 90% of the tryptophan in culture media overnight. In VNP20009- tnaCAB depleted tissue culture media, MDA-MB-468 breast cancer cells were unable to divide, while those treated with media exposed only to VNP20009 continued cell division. Treatment with the TNase products indole, pyruvate and ammonia did not have an effect on tumor cell growth. These data suggest that TNase depletion of tryptophan may augment the antitumor activity of VNP20009 and may further limit production of immunosuppresive kynurenine.
Nancy Chavez (Undergraduate), CSU, Fresno - Biology
Examining Angiogenic Biomarkers in Aggressive Tumor Types of Thyroid Cancer
Thyroid cancer is the most common endocrine malignancy in the United States with over 52,000 new cases diagnosed annually and over 2,100 deaths. The second most common type of well-differentiated thyroid cancer is the follicular variant of papillary thyroid cancer (FVPTC). A critical issue in the pathology field involves refining which lesions of FVPTC are benign and those that have potential towards malignancy and metastasis. Our goal is to evaluate whether thyroid tumors with elevated expression of angiogenic (blood vessel-forming) biomarkers correlate with clinical features of aggressiveness. We recruited a test cohort of 20 archival FVPTC tumor tissue specimens and optimized tissue recovery for laser microdissection (LCM). LCM was performed with multiple cuts between 1500-2000 micrometers to separate tumor tissue from adjacent normal control tissue. From this micro-dissected material, RNA was extracted for downstream semi-quantitative RT-PCR analyses of common angiogenic factor gene expression. Preliminary results demonstrate the difficulty in maximizing reliable and quantitative differences from archival tissue when comparing tumor versus control via gel electrophoresis. Therefore, we have moved to a SYBR green qPCR-based strategy that allows for enhanced quantitative analysis of genetic material and avoids utilizing gel electrophoresis. Our hope is to integrate pathologic findings with molecular validation for improved predictive clinical utility.
Armenuhe Terzian (Undergraduate), CSU, Northridge - Chemistry/Biochemistry
Bisiminophenanthrene-based anti-tumor agents: exploiting active transport processes to target cancer
Much research is being devoted to develop therapeutics that better target cancer cells and avoid the familiar complications of chemotherapy. The Kelson group is currently developing bis(R-imino)phenanthrene (BipR) anti-tumor agents designed to mimic nutrients actively harvested by tumor cells and induce apoptosis once inside cells. BipR agents with simple R groups such as phenyl, benzyl, 2-pyridylmethyl, and n-butyl are surprisingly toxic against MDA-MB-231, MDA-MB-468, and MDF-7 breast cancer cell lines with EC50s ranging from 105 to 840 nM. These toxicities are modestly inhibited by added spermine and tetrapropylammonium chloride suggesting active transport of BipRs by polyamine and organic cation transporters, respectively. BipR agents have also been prepared with amino acid and glucosamine R groups to target the corresponding transporters. These products indeed exhibit greater toxicities (EC50s ranging from 34 to 305 nM) than those with simple R groups. The potency of Bip(glucosamine) is inhibited by 2-deoxy-D-glucose as well as by glucose supplementation of the growth media suggesting assisted cell entry through sugar transporters. Similarly, the toxicities of amino acid based BipRs are inhibited by growth media supplementation with the corresponding amino acids suggesting assisted entry. Overall, the BipR toxicities appear to validate the strategy of exploiting active transport processes to attack and perhaps target tumor cells.
Logan Kasper (Undergraduate), CSU, Northridge - Chemistry/Biochemistry
Novel Small Molecules Inhibit the Cell Cycle Through GSK3 Signaling Pathway
A successful cancer therapeutic agent should induce cell cycle arrest in cancerous cells and have potential as therapeutics in the clinical setting. Niclosamide is an oral antihelminthic drug used to treat patients with parasitic infections. However, studies have indicated that niclosamide may be applicable to treat diseases other than parasites, such as cancer. New compounds synthesized in the de Lijser Lab have also shown the potential as a cancer therapeutic. The goal of our lab is to determine their effects on the cell cycle and determine their mechanism. The Wnt Pathway is known to regulate the cell cycle and has been of interest in the development of cancer due to the dysregulation of β-catenin. When β-catenin is not required by the cell, it is continuously degraded by the protein complex composed of CK1α, GSK3, Axin, and APC. Phosphorylation of β-catenin by GSK3 allows it to become ubiquitinated and marked for degradation. Our research has found that niclosamide downregulates GSK3 presence and activity. This indicates that Niclosamide may have broad reactivity and therefore may not be a proper candidate as a therapeutic agent. However, compounds synthesized in Dr. de Lijser’s laboratory may be a more effective alternative.