Eric Aguilar, Poster #44 (CSU Fresno)
Structure of a Therapeutic Antibody in Complex with MUC16 Reveals a Conformational Epitope Influenced by Antigen Glycosylation
The integral membrane glycoprotein Mucin-16 (MUC16) has emerged as an important cancer antigen that displays a high degree of tumor selectivity. MUC16 is overexpressed and involved in tumorigenesis in several malignancies, including pancreatic and ovarian cancer. The role of MUC16 in cancer progression is complex, and provides multiple points for therapeutic intervention. However, despite clinical interest in MUC16 as an immunotherapy target, surprisingly little is known regarding how antibodies bind the protein. Here we report the humanization, epitope mapping and structure determination of a MUC16 specific therapeutic antibody. The antibody was humanized using a germline complementary determining region (CDR) loop grafting approach, and produced by transient transfection in Chinese hamster ovary (CHO) cells. The humanized and murine antibodies displayed nearly identical affinity to a recombinant MUC16 SEA (Sperm protein, Enterokinase and Agrin) domain as measured by enzyme linked-immunosorbent assay (ELISA) and surface plasmon resonance (SPR). High resolution x-ray structures of the antibodies indicated no significant changes associated with humanization. Initial epitope mapping using an ELISA and overlapping MUC16 constructs indicated that the antibody epitope was localized to an SEA domain and was non-linear and conformational in nature. A more detailed epitope mapping study carried out using hydrogen- deuterium exchange mass-spectrometry revealed five regions on the SEA domain that resulted in reduced deuteration when antibody was bound to antigen. These results were confirmed by x-ray structures of the murine and humanized antibody in complex with the SEA domain. The structures reveled a complex, non-linear structural epitope with a long -hairpin forming the center of the interaction. Finally, a fully glycosylated recombinant SEA domain was produced by transient transfection in CHO cells and used to assess the role of MUC16 glycosylation on antibody binding. Surprisingly, the densely glycosylated SEA domain bound the antibody with approximately 2-fold higher affinity compared to the unglycosylated domain, suggesting a role for antigen glycosylation in mediating antibody binding. The results presented here represent the first structural characterization of an antibody in complex with MUC16 and reveal a complex, non-linear epitope influenced by glycosylation. These finding will help to accelerate clinical development of this promising therapeutic agent.
Elene Albekioni, Poster #14 (CSU San Diego)
Kinetic and Structural Properties of Human Isocitrate Dehydrogenase 1
Isocitrate dehydrogenase 1 (IDH1) is a cytosolic enzyme responsible for maintaining the redox environment and α-ketoglutarate (AKG) pools in cells. At physiological pH, IDH1 forms catalytically competent homodimers that convert isocitrate (ICT) to AKG in an NADP+ -dependent oxidative decarboxylation reaction. Mutations in IDH1 are associated with multiple tumor types due to accumulation of oncometabolite D-2-hydroxyglutarate (D2HG). However, it is still unclear what is the mechanism of catalysis and what kind of conformational changes are associated with it. We adopted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) technique to directly analyze structural modifications by allowing active exchange between backbone amide hydrogen atoms and surrounding D2O. Even though HDX-MS is limited to peptide resolution, comparing the fluctuations in deuterium uptake by holo and apo wild type IDH1 gives significant information regarding residues involved in substrate coordination. We used spectroscopic tools to measure kinetic activity of IDH1 by monitoring the rate of NADPH formation and consumption in reactions. We compared catalytic efficiency of wild type, important mutants, and wt/mutant heterodimer IDH1 since heterodimerization likely occurs in IDH1 induced tumor cells. Understanding how rates of the reactions compare in the IDH1 homodimer and heterodimer complexes together with HDX-MS results allow us to better understand the molecular mechanisms of IDH1 catalysis in tumors.
Albert Aquino, Poster #1 (CSU Northridge)
3D Tumorsphere Studies to Identify New Regulators of PDAC Progression
Pancreatic Ductal Adenocarcinoma (PDAC) is the most common form of pancreatic cancer with a 5 year survival of less than 10%. This is due to a lack of early diagnosis markers and chemotherapy resistance stemming from a dense microenvironment with stromal cells and extracellular matrix components. We use a 3D spheroid culture system in ultra-low attachment round bottom multi-well plates for high-content imaging of spheroid dynamics in response to pharmacological and environmental perturbations. PDAC cells with a mixed epithelial/mesenchymal morphology (e.g., PANC1) or fully mesenchymal morphology (e.g., MiaPaca2) formed loose aggregate spheroids. PDAC cells with a fully epithelial morphology (e.g., BxPC3) formed tight compact spheroids. The addition of mesenchymal stromal cells (MSCs) to form multicellular spheroids induced a strong compaction phenotype in PANC1 and MiaPaca2. Analysis of spheroid compaction and growth in response to a panel of exogenous factors further revealed that inhibition of the motor protein, dynein, prevents spheroid formation across PANC1, MiaPaca2, and BxPC3. Inhibition of glycolysis increases compaction of PANC1 and MiaPaca2 cell lines. Future studies will evaluate how treatments that alter PDAC spheroid dynamics affect changes in EMT and stemness gene expression signatures. Taken together, these studies demonstrate the utility of high-throughput 3D assays and uncover targeted treatments that may render PDAC more responsive to chemotherapies.
Melissa Aros, Poster #32 (CSU Northridge)
Investigating SLX4-Dependent Recruitment of Rad1-Rad10 to DNA Double Strand Breaks During DNA Repair in Dividing Cells
Once DNA damage is introduced into a cell, it triggers checkpoint arrest and inhibits replication initiation as a result. Another phenomenon induced by DNA damage is the phosphorylation of Slx4, which facilitates a counteracting pathway we are studying called checkpoint signal dampening. It promotes homologous recombination, thereby aiding in DNA Double-strand break repair. Our project focuses on whether the phosphorylation of Slx4 affects recruitment of Rad1-Rad10 in double-strand break repair sites in S. cerevisiae in either Synthesis-Dependent Strand Annealing (SDSA) or Single-strand Annealing (SSA). We hypothesize that checkpoint signal dampening leads to Slx4 phosphorylation, which plays a role in the recruitment of Rad1-Rad10 to double-strand breaks observed in the S phase of the cell cycle. To test this, we utilized an Slx4 phosphorylation mutant (slx4-7MUT) that prevents the activation of checkpoint signal dampening and allows us to determine whether fluorescently-labeled Rad10 (Rad10-YFP) is recruited to DNA damage sites when Slx4 is unable to be phosphorylated. We have noticed in our fluorescence microscopy and flow cytometry results that in120 minute release time-point, the percentage of co-localized foci in the S phase is statistically similar between the induced and uninduced samples. This suggests Slx4 checkpoint signal dampening plays a role in Rad1-Rad10 recruitment to double strand break sites in DNA repair at certain timeframe.
Natan Barros, Poster #35 (Terasaki Institute)
Nanoengineered Injectable Drug-eluting Hydrogel as a New Generation of Transcatheter Arterial Embolization to Treat Liver Cancer
Hepatocellular carcinoma (HCC), the most common type of liver cancer, is often detected at advanced stages where treatment options are limited. While systemic chemotherapy has been the cornerstone of cancer treatment, the inability to achieve uniform drug delivery to tumors, collateral toxicity to the non-cancerous liver, and systemic side effects have limited progress in developing novel therapies for liver cancer. Here, we combine transarterial chemoembolization (TACE) with immunotherapy to enhance the antitumor immune response. To achieve this goal, we mixed doxorubicin (DOX) and α-PD1 within our developed injectable shear-thinning hydrogel (STH). The release profile revealed that lower amounts of nanosilicate led to higher pH-responsiveness capable of achieving a localized and sustained release of DOX in an acidic tumor microenvironment (pH 5), while a high release of α-PD1 was achieved at physiological pH (7.4). Furthermore, DOX-loaded STHs could only efficiently target liver cancer cells in vitro via cell death and growth restriction when at low concentrations of nanosilicate or high concentrations of DOX, once more evidencing the need for combining with immunotherapy. Moreover, in vitro studies proved that α-PD1 released from low and medium nanosilicate STHs were still functional for complexation with α-PDL1. These results highlight the advantages and potential of using drug-eluting STH to successfully co-deliver immunotherapies for liver cancer therapy through TACE.
Ryan Boggess, Poster #39 (CSU Northridge)
Investigating the Ability of Metformin to Scavenge for Methylglyoxal and Impact Downstream Biological Effects
Methylglyoxal is a highly reactive ⍺-oxoaldehyde that arises as a byproduct of glycolysis, lipid metabolism, and amino acid metabolism. Recent evidence has shown that methylglyoxal reacts with protein, lipid and DNA to form advanced glycation end-products (AGEs). Metformin is a commonly prescribed drug for diabetics and evidence has shown it to lower the relative risk for a variety of cancers in metformin users relative to non-diabetic individuals. The biguanide pharmacophore of metformin has been found to react with methylglyoxal forming an imidazolinone ring. My work is focused on investigating metformin’s ability to scavenge methylglyoxal resulting in diminished protein and DNA-AGEs and decreased mutations. I have synthesized and purified methylglyoxal from pyruvic dimethylacetal through an acid-catalyzed reflux reaction followed by a fractional distillation with two of five fractions containing pure methylglyoxal as determined through 1H-NMR analysis. The reaction between methylglyoxal and metformin has been briefly investigated through semi-quantitative HPLC resulting in a 50% decrease in metformin peak area. Metformin’s effect on free methylglyoxal concentration has also been investigated through an Abcam methylglyoxal assay indicating a 70% decrease in methylglyoxal concentration by the fourth day. Caco-2 cells have been grown with and without metformin in the media. Glyoxylase inhibitors and free methylglyoxal will be utilized to observe metformin’s protective effect.
Jane Cox, Poster #19 (CSU Northridge)
The Role of Lcn2 in Mediating in Global Gene Expression Changes in Breast Cancer
Cancer is one of the leading causes of death worldwide. Among women, breast cancer has one of the highest cancer-related death rates, second to only lung cancer. The metastasis of breast cancer contributes significantly to the cause of breast cancer patient death. Previous research from my lab determined the secreted factor Lcn2, or Lipocalin-2, to be highly up-regulated in metastatic breast cancer cells. We created shRNA derivatives to knockdown Lcn2 in the metastatic breast cancer cell line Py230. We validated the knockdown with WesternBlot, qPCR, and ELISA, which all showed significantly decreased levels of Lcn2 in the knockdown line when compared to the control. I have performed a series of qPCRs looking at 34 different known cancer related genes on the scramble and Lcn2 knockdown derivative cell lines to investigate the possible effects of Lcn2’s absence. I found that two of the 34 have a significant gene expression change, namely Serpine1 and TNF, which was an increase in gene expression. My project’s focus is to investigate the effect of Lcn2 on other cancer related genes in metastatic and triple negative breast cancers. My goal is to perform the same assays on the TNBC cell line SUM149 to analyze with my Py230 data in order to determine which genes are Lcn2-dependent and how their gene expression is affected.
Luis Jesus Cruz, Poster # 29 (CSU San Diego)
Role of macrophages in the development of ovarian cancer stem-like cells
Ovarian cancer is a poorly understood disease that has a 75% death rate when found after metastasis. Drug resistance and tumor recurrence are likely due to cancer stem-like cells (CSCs), unlike bulk tumor cells, can evade chemotherapy and induce relapse. However, it is still unclear on how CSCs facilitate disease relapse and what role the tumor microenvironment (TME) plays in this process. Preliminary data shows that secreted TWEAK and its receptor Fn14 are overexpressed in ovarian tumors which increases in the presence of chemotherapy, TWEAK is a strong inducer of stem cell activity, and TWEAK enhances survival of CSCs. Publicly available clinical data from human ovarian tumor found that TWEAK mRNA was primarily observed in a subset of infiltrating immune cells known as tumor associated macrophages (TAMs). Since TAMs seem to be the main source of soluble TWEAK and the fact that cytotoxic chemotherapy can enrich for different TAM populations, we propose that TAMs might be responsible for production of TWEAK following chemotherapy, and that it supports CSC populations and relapse potential. Preliminary targeting of TWEAK with a small molecule inhibitor showed prolonged remission in mouse models of ovarian cancer. Therefore, we will knockout Fn14 on ovarian cancer cells to see if CSCs are dependent on TWEAK-Fn14 signaling for relapse and we will investigate the role of TAMs in supporting CSCs while identifying the specific TAM population responsible for TWEAK production.
Menekse Ermis, Poster #33 (Terasaki Institute)
Mimicking Desmoplastic Pancreatic Adenocarcinoma Matrix In Vitro Using 3D Cancer-Fibroblast Spheroids Embedded HAMA-GelMA Hydrogels
The tumor microenvironment comprises complex biological and physical factors such as extracellular matrix, cell-cell interactions, tissue mechanical properties, and growth factor-related signals. In pancreatic ductal adenocarcinoma (PDAC), which exhibits marked desmoplastic features, stromal tissue plays an important role not only in physical properties but also in pancreatic cancer progression and therapeutic responsiveness. So far, no established model could fully recapitulate all these components to resemble and understand the progression of pancreatic tumors. Here, two major components in desmoplastic pancreatic matrices, hyaluronic acid (HA)- and collagen-based hydrogels, are engineered to provide matrices for tumor spheroids composed of pancreatic cancer and cancer-associated fibroblasts (CAF). Analysis of shape profiles and cancer-CAF co-localization reveals that stromal cells are required for more compact tissue formation. Higher expression levels of markers associated with epithelial to mesenchymal transition, mechanotransduction, and progression are observed for spheroids cultured in hyper desmoplastic matrix-mimicking hydrogels or in desmoplastic matrix-mimicking hydrogels with the presence of transforming growth factor-β1 (TGF-β1). The proposed multicellular pancreatic tumor model, in combination with proper mechanical properties and TGF-β1 supplement, makes strides in developing advanced models for resembling and monitoring the progression of pancreatic tumors.
Joshua Gamez, Poster #24 (CSU Northridge)
The Role of Lcn2 In Mediating Breast Cancer Metastasis In Premetastatic Lung
The premetastatic niche is a cellular composition of tissues and organs that lay the foundation for tumors to metastasize and understanding this priming cascade is important for understanding the basis of metastasis. Previous data has shown that secreted factors from metastatic Py230 cells are able to remodel the lung microenvironment and support tumor proliferation as opposed to non-metastatic Py8119 cells. Immunoblot and ELISA data has shown that the secreted factor Lcn2 exhibits selective expression in Py230 cells and circulating levels of Lcn2 are increased when treated with Py230 conditioned media. An in-vivo experiment was done where mice were injected with conditioned media from Py230 & Py8119 cells and RNA-sequencing was done on the CM-educated lung tissues. Gene expression analysis was done to generate gene ontologies that represent the lung transcripts that are both induced and suppressed by the presence of Lcn2 in Py230 cells. Clinical outcome of these enriched genes was observed to predict patient survival. From this, shRNA derivatives were created to knockdown Lcn2 in these Py230 cells. These knockdowns have been characterized with subsequent qPCR, ELISA, and Western data. An in-vivo experiment has been devised to collect tissues that have been treated with conditioned media from this knockdown line. Global & single-cell omics will allow us to dive deeper into the molecular changes that may be happening within the Lcn2-dependent premetastatic microenvironment.
Amir Gerami, Poster #3 (Western University Health Sciences)
LCN2 Function in Triple Negative Breast Cancer Spheroids/Organoids
Breast cancer (BC) is among one of the most common cancers in the world. Unfortunately, those with metastatic BC tumors have significantly less chance of survival. Among different BC subtypes, the triple negative breast cancer (TNBC) subtype has been found to be difficult to treat and has a higher mortality rate. Transcriptome analysis of our metastatic TNBC line, Py230, revealed a lipocalin 2 (LCN2) axis that is selectively expressed, predicts poor BC patient survival. Other research have reported that LCN2 promotes BC progression and metastasis. We sought to further study various roles of the LCN2 in metastatic TNBC by hypothesizing that TNBC-derived LCN2 mediates invasion and premetastatic lung reprogramming to support metastasis. Recent research have shown promising evidence of using organoids/spheroids models for precise study of BC and drug discovery. Hence, we cultured the Py230 cells both in 3D suspension and on Matrigel domes which led us to engineer tumor spheroids and organoids respectively. Next, the size and shape of the spheroids/organoids were analyzed and contrasted between the LCN2 KD and control groups. Our preliminary in vitro findings suggest LCN2 mediates TNBC organoid invasion/protrusion and enhances inter-organoid bridging. Moreover, LCN2 KD enhances compactness in TNBC spheroids. These results establish the role of LCN2 in TNBC tumor morphology which could be a promising therapeutic target for blocking BC progression and metastasis.
Jocelyne Milke, Poster #22 (CSU San Francisco)
Functional Assays of Breast Cancer Cell Lines from Representative Racial Categories
Black and Hispanic women are more likely to die from breast cancer than White women. Breast cancer is divided into the molecular subtypes TNBC, HER2 positive, and luminal. For this study we hypothesized that breast cancer cell lines from minority women exhibit worse phenotype, characterized by enhanced migration, invasion, and inflamed macrophages and endothelial cells. We conducted migration and invasion assays on nine breast cancer cell lines, from representative racial categories, using Boyden chambers and Matrigel as the extracellular matrix. Microscopy imaging was used for quantification. Co-culture assays of macrophages and cancer lines and trans-endothelial migration assays will be analyzed using flow cytometry. We found that cell lines from Black women with TNBC status were 21% more migratory but 2% less invasive than the cell line with TNBC status from White women. TNBC cell lines were 59% more migratory than HER2 and 69% more migratory than luminal cell lines. We expect significantly higher levels of inflammatory cytokines in minority cell lines. Our studies use biologically representative cell lines from minority groups which have been understudied. Our results identify functionally significant differences dependent on racial category despite the same molecular subtype and thus help set the groundwork for improved personalized medicines that address racial disparities in breast cancer.
Osvaldo Moreno, Poster #37 (CSU San Francisco)
Determining the Role of vGPCR in Modulating Host Cell Transcription and Metabolism
Kaposi Sarcoma-associated Herpesvirus (KSHV) is a small etiologic agent that can induce angiogenesis in endothelial cells, and lead to Kaposi Sarcoma (KS). KSHV has two life cycles: the latent, a more dormant stage of infection, and the lytic stage, in which virus is actively produced. Viral G Protein-Coupled Receptor (vGPCR) is a lytic signaling protein that has been connected to modulating host cell transcription and paracrine signaling. Our previous work identified the host cell metabolism to be crucial for KSHV infection, but the mechanism by which vGPCR modulates cellular metabolism has yet to be discovered. We hypothesize that vGPCR signaling dramatically alters the host transcriptome and metabolism. To test our hypothesis, we have successfully overexpressed vGPCR protein in mammalian cells, which was confirmed through western blots and immunofluorescence assays. We performed RNA sequencing analysis to obtain a host cell transcriptomic profile for vGPCR-expressing cells compared to empty vector expressing cells. The RNA-Seq analysis identified the upregulation of cyclooxygenase-2(COX-2), which was validated via qPCR. We show vGPCR can upregulate COX-2, an enzyme that is implicated in modulating glycolysis, and required for viral lytic replication. Therefore, these experiments will elucidate the mechanism by which vGPCR modulates metabolic gene expression and cellular metabolism, helping lay the foundation to develop antiviral therapeutics.
Ashfeen Nawar, Poster #4 (CSU San Diego)
Purification and kinetic investigation of Human DNA Polymerase ε
In eukaryotic replication, DNA polymerase epsilon (Pol ε) is in charge of leading strand synthesis. The catalytic polymerase domain is responsible for correct nucleotide incorporation whereas the exonuclease domain excises misincorporated nucleotides. Importantly, mutations in Pol ε have been implicated in a variety of cancers, including uterine, colorectal, skin, and stomach. These tumor-driving mutants have not yet been well characterized. Using pre-steady-state kinetics, we will characterize the incorporation, misincorporation, and excision by wild type (WT) and mutant Pol ε. Here, by using a truncated active form of the enzyme, we will first optimize the heterologous expression and purification of WT Polε. To improve protein activity and yields, we tested different buffers and chromatography methods to obtain protein of high purity and activity. Then, to investigate the correct incorporation of single nucleotide in D-2 DNA substrate by WT Pol ε, we performed burst assay experiments by using a rapid chemical quench. We observed an initial burst of product formation associated with single enzyme turnover followed by a slower linear phase corresponding to multiple enzyme turnovers in the steady-state, typical of DNA polymerases. We plan to compare the observed rates with a R665W Pol ε mutant found in the polymerase domain to determine if the fidelity of Polε is affected. Ultimately, this will help us understand the factors that drive genomic instability in human cancer.
Cynthia Petrossian, Poster #8 (CSU Northridge)
Investigating the effects of manipulating downstream targets of two master regulators of intestinal stem cells in the Drosophila melanogaster posterior midgut
The integrity of organs and tissues relies on adult stem cells (ASCs), which can divide asymmetrically, giving rise to a new copy of themselves and a sister cell that differentiates along a cell lineage. Many internal and external signals control ASC division and differentiation, relayed via master regulator (MR) genes, which are pleiotropic genes that control multiple downstream targets. While the effect of manipulating MRs on ASCs has been well characterized, and several of their potential downstream targets have been identified, validation of these targets and a mechanistic explanation of how they function within ASCs is still largely lacking. We use Drosophila melanogaster intestinal stem cells as a model system to study ASC biology and regulation, and the transcription factors Esg and STAT, two well-known MR genes in these cells. Previous work from our lab has validated a series of downstream targets of Esg and STAT, and we are now investigating the effects that manipulating two such targets, Foxo and Indy, may have on turnover and morphological features of intestinal stem cells. Our preliminary data suggest nonlinearities in the behavior of pathways downstream of MRs, since manipulating Foxo and Indy did not phenocopy the manipulation of the corresponding MRs. Understanding such nonlinearities in stem cell behavior may be critical for managing complexities in the response of cancers to treatments targeting cancer stem cells.
Janelli Pineda, Poster #20 (CSU Northridge)
Construction of Yen1-RFP and Investigation of Yen1-RFP Recruitment in DNA Double strand Breaks at Anaphase Bridges
Double-strand breaks(DSBs) in DNA arise from a variety of sources that may impact and stall replication, leading to mutations in DNA that cause biological defects. DSB repair mechanisms are conserved across species and pathway choice depends on several factors, including cell-cycle phase. In this work, we are investigating whether a red fluorescently-tagged endonuclease, (Yen1-RFP), localizes to DSBs found at anaphase bridges. The first phase of this project entails the construction of a plasmid containing Yen1-RFP that was subcloned from a Yen1-GFP plasmid and transformation of a yeast strain with the Yen1-RFP fragment.
To achieve this goal, the RFP gene from pNEB30 was swapped with the GFP gene in the Yen1-GFP plasmid using standard cloning techniques including PCR and restriction digestion. After construction of the Yen1-RFP plasmid, the Yen1-RFP fragment was isolated from the plasmid and transformed into a strain of S. cerevisiae containing wild-type YEN1 to create a recombinant Yen1-RFP strain. This strain will be screened by PCR, DNA sequencing, and fluorescence microscopy to confirm that the Yen1-RFP gene was successfully incorporated. The Yen1-RFP gene will be combined with other genetic features to eventually create the strains needed to investigate the localization of Yen1 to anaphase bridges by fluorescence microscopy. The experiments will provide insights regarding the role of Yen1 in DSB repair and how DNA repair pathways are cancer protective for humans.
Sabrina Rabaya, Poster #40 (CSU Los Angeles)
Impact of Adenylate Kinase Expression Levels on Lung Adenocarcinoma Viability
ATP homeostasis is a central component of cellular metabolism. This is exemplified in the observation that the bioenergetic processes responsible for maintaining ATP levels are dysregulated in many types of diseased cells. Cancer cells, for example, rely on increased energy levels in order to sustain their proliferation. Adenylate Kinases (AK) are enzymes responsible for the maintenance of ATP, ADP, and AMP levels in different areas of the cell. There are nine AKs found in the human body. AK1 is found in the cytosol and AK2-4 are found in the mitochondria. The remaining localizations of AK5-9 are found in various components of the cell. Previous work identified significant changes in AK expression levels within lung adenocarcinoma compared to normal lung cells. This would suggest that AKs play a crucial role in the maintenance of these cancer cells. However, a thorough analysis of the impacts of AK expression in lung cancer has not been conducted. Therefore, this project aims to identify the effects of silencing the expression of each AK in lung cancer cells. We will achieve this goal by performing a small interfering RNA (siRNA) screen targeting each AK in a lung cancer cell model. We hypothesize that the silencing of adenylate kinases will negatively impact the viability of lung adenocarcinoma cells.
Leticia Reque, Poster #28 (CSU Northridge)
Investigating the effect of Glo1 inhibition of Nrf2 translocation, elevated AKR expression and the glutathione biosynthetic response in cancer cells
Breast cancer and prostate cancer are the two of the most commonly diagnosed cancers in the developed world. In breast tumor biopsies, Glyoxalase 1 and 2 (Glo 1/2) are over-expressed relative to adjacent tissue. In prostate tumors, high levels of Glo 1 expression correlates with poor prognosis. The Glo 1/2 system is critical for the detoxification of metabolically derived dicarbonyl species such as methylglyoxal and glyoxal. The inhibition of Glo 1 by a glutathione analogue, results in cell death in a panel of breast, prostate, and ovarian cancer cell lines (EC50 = 9.0-29 uM). We used qRT-PCR and observed 2- to 3-fold increases in AKR1C1 in the MCF-7 and MDA-MB-231 cell lines and 2-fold increase in AKR1C3 in the MCF-7 and 22Rv1 cell lines. We observed the increased Nrf2 translocation in the MCF-7, MDA-MB-231, and 22Rv1. Taken together, these findings corroborate that elevated MG in the cell results in translocation of Nrf2 and elevated AKR expression. Additionally, we used western blotting to observe the enrichment of GCL and GS in these cell lines. We observed an increase in GCL protein levels in the MDA-231 and LNCaP cell lines, as well as an increase in GS protein levels in the MCF-7, DU145, and 22Rv1 cell lines. Taken together, these results suggest a potential role of GCL and GS in response to Glo1 inhibition.
Sabrina Sharma, Poster #41 (CSU Northridge)
Iminodiazaphenanthrene-based “Trojan horse” anti-tumor agents
Targeted chemotherapies are of recent interest for their promise of improved toxicity against tumors without the familiar complications of chemotherapy. The Kelson group is currently exploring the preparation and toxicity of iminodiazaphenanthrene-based “Trojan horse” anti-tumor agents that mimic nutrients actively harvested by cancer cells. Specifically, iminodiazaphenanthrenes (IdpRs) bearing simple benzyl, 2-pyridylmethyl, and n-butyl R groups are surprisingly toxic against MDA-MB-231, MDA-MB-468, and MDF-7 breast cancer cell lines with EC50s ranging from 100 to 850 nM. These toxicities are modestly inhibited by added spermine or tetrapropylammonium chloride suggesting some active transport of IdpRs by polyamine and organic cation transporters, respectively. IdpR agents with amino acid and glucosamine R groups exhibit greater toxicities (EC50s ranging from 35 to 300 nM). The toxicity of Idp(glucosamine) agents are inhibited by 2-deoxy-D-glucose, and the potency of some amino acid bearing IdpRs are suppressed by media supplementation with the corresponding amino acids as expected for cell entry by nutrient transport mechanisms. Interestingly, the triple-negative MDA-MB-231 cell line is especially sensitive to IdpRs, and some amino acid derived IdpRs are particularly toxic toward MDA-MB-468 cells. This selectivity suggests that IdpRs could target specific cancers. Overall this work appears to validate the “Trojan horse” strategy and holds promise for future chemotherapies.
David Stachura, Poster #10 (CSU Chico)
ccl44 is Essential for Normal Embryonic Zebrafish Hematopoiesis
Hematopoiesis (blood formation) must occur throughout the life of an organism. Capable of self-renewal and differentiation into daughter cells, hematopoietic stem and progenitor cells (HSPCs) differentiate into all the mature blood cells in the body. Studying the molecular pathways responsible for this differentiation allows better understanding and treatment of a multitude of blood diseases, which arise from defects in these processes. The zebrafish (Danio rerio) is an ideal model organism to use for the study of hematopoiesis because their blood development is similar to humans and they have high fecundity. In addition, the embryos are transparent and develop outside the body making manipulation and observation easier. Through RNA sequencing, our laboratory has identified the top 100 genes integral to HSPC maintenance identified in zebrafish supportive stromal cells. One such gene was chemokine ligand 44, ccl44. Knockdown with morpholinos and ablation with CRISPR/Cas9 showed a reduction in fluorescently labeled erythroid and myeloid cells which were then successfully rescued with co-injection of mRNAs. Many phenotypic defects were also seen in the mutated embryos, including shortened tail length and spinal curvature, consistent with a defect in mesoderm patterning. Elucidating the role of ccl44 in hematopoiesis should help inform us about the evolution of the vertebrate hematopoietic system and could have clinical importance for the treatment of human blood diseases.
David Stachura, Poster #11 (CSU Chico)
mustn1a is essential for normal vertebrate hematopoiesis
Hematopoiesis is the differentiation of hematopoietic stem cells (HSCs) into a multitude of mature blood cells. This process is vital in the early developing embryo and is sustained throughout an organism’s lifetime. Our laboratory uses the Danio rerio (zebrafish) model to explore hematopoietic development, and has successfully identified 447 genes likely important for these processes. Of these genes, mustn1a had high expression in areas of hematopoiesis. We hypothesized that elucidating the role of mustn1a in zebrafish hematopoiesis would better our understanding of the evolutionary history behind this process in all vertebrates. To do this, expression of the gene was knocked down in developing zebrafish embryos using a mustn1a-specific morpholino (MO) via microneedle injections at the single-cell-stage. Knockdown resulted in a reduction of neutrophils, erythrocytes, and thrombocytes in developing transgenic zebrafish. These results were validated by mRNA rescue. mustn1a reduction also resulted in body axis deformities in morphants. Previous studies indicated that this gene is important for condensation of mesenchyme to form cartilage and bone, and our observations give us reason to believe that it is also involved in mesodermal differentiation. In essence, mustn1a is important for fish hematopoiesis as well as formation of mesodermal structures. Further studies will be performed to see the importance of this gene and its orthologs during mammalian hematopoietic development.
David Stachura, Poster #12 (CSU Chico)
rapunzel5 is necessary for normal hematopoietic development in zebrafish
The molecular mechanisms regulating the highly complex process of hematopoiesis in vertebrates is still enigmatic. This system begins with the controlled differentiation of adult hematopoietic stem and progenitor cells (HSPCs), which can proliferate and generate all types of mature blood cells. Identifying the underlying factors and mechanisms that allow HSPCs to differentiate and proliferate is an essential issue in stem cell biology and tissue homeostasis; disruptions in these processes can cause severe diseases. A transcriptomic screen of hematopoietic-supportive zebrafish stromal tissues identified rapunzel5 (rpz5) was likely involved in vertebrate hematopoiesis. We performed loss-of-function experiments in zebrafish embryos at the one-cell-stage with morpholinos to determine if blood cell production was affected by rpz5. rpz5 knockdown resulted in reduced amounts of red blood cells, myeloid cells, and thrombocytes, and adding back exogenous rpz5 rescued these deficiencies. Further analysis with methylcellulose assays indicated that there was also a significant reduction in HSPCs following rpz5 reduction. Together, these findings suggest that zebrafish rpz5 is essential for normal formation, differentiation, and proliferation of HSPCs, specifically down the erythroid and myeloid pathway. Fully understanding the roles of novel genes such as rpz5 is essential for understanding the evolution of vertebrate hematopoiesis and for treating hematological diseases in the future.
Kyaw Hsu Thway, Poster #27 (CSU Northridge)
Analysis of PEAK1/VCL cooperativity in MSC mechanotransduction and MSC-driven HER2-positive breast cancer progression
Mesenchymal stromal cells (MSCs) drive HER2+ BC progression via the secretion of tumorigenic factors and by serving as precursors to cancer-associated fibroblasts (CAF). Our group has characterized a novel SNAI2/PEAK1/activin-A signaling axis in MSCs/CAFs that promotes HER2+ brain metastasis and targeted therapy resistance. Since PEAK1 is a core component of integrin adhesion complexes (IACs) that governs extracellular matrix and growth factor crosstalk, we will study how PEAK1 may enable MSCs to control their cell state dynamics and tumorigenic potential. PEAK1 knockdown in MSCs led to an alternation of the IAC proteome and bioinformatic analyses of PEAK1-dependent MSC IAC revealed a clinically relevant role for Vinculin (VCL) and upstream signaling via NIMA related kinase 2 (NEK2). We observed that blockade of NEK2 abolished the detection of phosphorylation on Ser/Thr residues in VCL. Since PEAK1 and VCL roles in mechanotransduction were established, we quantified gene expression for a panel of PEAK1-related genes in mouse and human MSCs on substrates ranging in elastic moduli (0.2-64kPa). We observed that VCL expression in hMSCs under stiffer substrate conditions was elevated 4x more to cells under less stiff substrate. SNAI2, PEAK1, NEK2 and INHBA expression were not affected by substrate stiffness in both MSCs. We will further evaluate how NEK2 inhibition of VCL affects the expression of the PEAK1-related genes above which in turn drive HER2+ breast cancer progression.
Luke Tomaneng, Poster #30 (CSU Northridge)
Retinoic Acid Induced 14 Drives Pancreatic Cancer Cell Proliferation and Survival
90% of Pancreatic ductal adenocarcinoma (PDAC) patients die within 5 years after diagnosis. Hurdles facing PDAC patients and clinicians include early dissemination, lack of therapeutic targets and desmoplasia that drives chemotherapy resistance. We previously reported that pseudopodium-enriched (PDE) proteins PEAK1 (pseudopodium-enriched atypical kinase 1) and integrin α 1 (ITGA1) mediate gemcitabine resistance and metastasis in PDAC. By mining the Cancer BioPortal and Human Cell Map databases, we have identified the PDE protein Retinoic Acid Induced 14 (RAI14) as a potential mediator of PEAK1- and/or ITGA1-driven PDAC progression. To determine the role of RAI14 in PDAC cells, we made RAI14 knockdown and CRISPR knockout (KO) lines. In vitro, these derivatives were less motile, less proliferative and more sensitive to mitotic progression inhibitors (e.g., volasertib). To find signaling pathways and cell states in PDAC that are influenced by RAI14, we used cyclic immunofluorescence (CycIF) to spatially profile the proteome/phosphoproteome of RAI14 KO PDAC cells relative to wild type controls. Using single-cell analysis and bioinformatics methods, we found that RAI14 impinges upon tumor suppression and dysregulates survival pathways. Together with ongoing in vivo results, these studies uncover a novel control hub of PDAC progression and targetable upstream and downstream pathways that may lead to enhanced chemotherapy response, reduced tumor burden and improved patient outcomes.
Ranel Tuplano, Poster #38 (CSU Northridge)
Fibronectin Exposes a Targetable DHPS/SLC3A2 Vulnerability that can be Leveraged to Decrease Cytoplasmic Levels of eIF5A1/2 and Proliferation/Survival in TNBC
Less than 20% of breast cancer patients survive five years after metastasis as breast cancer ranks first among female-cancer related deaths. This emphasizes the need to investigate mechanisms that govern metastasis in triple-negative breast cancer (TNBC). Our lab previously found that eukaryotic initiation factor 5A 1/2, eIF5A1/2, regulates Pseudopodium-Enriched Atypical Kinase One, a pro-tumorigenic protein, which acts as a non-canonical switch for TGFβ, a hallmark to epithelial to mesenchymal transition. Our 2019 BBRC publication demonstrated that the pharmacological inhibition of eIF5A1/2 with GC7 blocked TGFβ/fibronectin induced metastasis to the lungs. Moreover, intratumoral heterogeneity is known to confer treatment resistance and increase metastasis. With this, we discovered a correlation between elevated solute carrier 3A2, SLC3A2, and decreased survival in breast cancer patients with amplified SMAD3, a tumor suppressive transcription factor in the canonical TGFβ pathway. DHPS inhibition alone could sequester SMAD3 into the nucleus. We also discovered inhibiting SLC3A2 or DHPS decreased TNBC spheroid growth. However, decreased effects were masked by fibronectin after an increase in cytoplasmic eIF5A1/2 levels were observed. Ongoing work aims to observe similar effects on SMAD3 localization and 3D formation and aggregation. All while elucidating on the intratumoral heterogeneity of TNBC in association of eIF5A 1/2 and SLC3A2 by utilizing cyclic immunofluorescence.
Matt Wallace, Poster #31 (CSU Northridge)
Proteomic Analysis of α1 Integrin-Dependent Adhesion Complexes in Pancreatic Cancer Identifies Targets for Chemotherapy Sensitization
Pancreatic ductal adenocarcinoma (PDAC) patients have an exceptionally high mortality rate and low median survival. Our lab previously reported that alpha one integrin (ITGA1) depletion inhibits pancreatic cancer metastasis and gemcitabine resistance. To identify therapeutic targets that may synergize with ITGA1 knockdown/inhibition, whole cell extracts (WCEs) and integrin adhesion complexes (IACs) were isolated from ITGA1 knockdown lines and analyzed by mass spectrometry. Of the 4553 and 1083 proteins identified within WCEs and IACs, 199 and 62 proteins changed upon ITGA1 knockdown in WCEs and IACs, respectively. ITGA1-induced IAC components that remained unchanged in WCEs were further analyzed by Cytoscape and notably showed convergence on KIFC1 and PLK1. Analysis of PDAC patient survival showed significantly reduced relapse-free and overall survival when both ITGA1 and PLK1 were elevated, which was not seen with KIFC1. This suggests that ITGA1 may recruit PLK1 into PDAC IACs to drive disease progression. Recent work focusing on ITGA1-PLK1 signaling in focal adhesion turnover revealed that both ITGA1 knockdown and PLK1 inhibition alone caused a significant increase in focal adhesion length. Interestingly, when both effects were present, focal adhesion lengths increased even further; suggesting an additive effect. Ongoing work aims to investigate the co-localization of well-known adhesion markers within IACs and the cooperative roles of ITGA1 and PLK1 in PDAC cell migration.