2020 Trainee Talks

10-minute presentation format

Merrick Courtney (graduate student), CSU Chico - Nutrition/Food Science

In Vitro Effect of Sulforaphane on Cytokine Secretions in Partial Breast Tumor Microenvironment

Triple negative breast cancer (TNBC) comprises 10–20% of breast cancer cases. It is particularly aggressive with limited and deleterious treatment options. Increasingly, research confirms that communication between cancer cell and neighboring macrophages promotes disease progression in part by secretion of cytokines that increase tumor cell proliferation, invasion, and metastasis. Sulforaphane (SFN) is a chemopreventive phytochemical found in cruciferous vegetables (broccoli) shown to alter cytokine secretion in macrophages and breast cancer cells grown in single culture. However, its effect in the tumor microenvironment remains unclear. This study investigates SFN influence on cytokine levels of TNBC cells and macrophages grown in coculture. We expect SFN to modify cytokine secretions in single vs coculture, suggesting SFN may disrupt vital cell-cell signaling. METHODS TNBC cells (MDA-MB-231) were grown in transwell plates with and without macrophages (THP-1 cells differentiated with PMA). Cell cultures (n = 3) were treated with either 15 µM SFN, DMSO (vehicle-control), or a non-treatment control. Cytokine levels were evaluated in media at 24 and 48 hours after treatment using BioPlex 2000 assay. RESULTS Preliminary analysis reveals that cytokine levels differed in cell culture media between treatment groups after 24 and 48 hours of treatment. These results may help identify mechanisms underlying SFNs chemopreventive function and inform new treatment strategies.

Francesca Sanchez (graduate student), CSU Northridge - Biology

Identification and Analysis of Breast Cancer Cell-Derived LCN2-Dependent Changes in the Premetastatic Lung Transcriptome

Metastasis is responsible for the majority of breast cancer-related deaths. The survival of cancer cells within distant tissues requires that these microenvironments acquire tumor-supportive characteristics. Recently the Kelber lab has established a tumor cell-free syngeneic in vivo breast cancer model for characterizing tumor cell secretome-mediated reprogramming of premetastatic tissues, where we reported that secretomes from metastatic breast cancer cells (BCCs), enriched for a prognostically unfavorable lipocalin 2 (LCN2) axis, induced anti-inflammatory MSC actions and a tumor-supportive premetastatic lung. In order to further elucidate how breast cancer cell secretomes remodel the premetastatic environment, lung tissue from mice were educated with condition media (CM) containing secretomes form metastatic breast cancer cells. RNA-sequencing was then performed on the CM-educated lungs, and further analyzed using Cufflinks. Bioinformatic data on the lungs that were educated with metastatic cancer cell secretomes revealed an upregulation in genes such as CCL19, a critical regulator of the induction of T cell activation, and WDYHV1, and a downregulation in genes such as DNAJB9 and SPOCK2 – all were shown to be significantly correlated with poor patient survival using cBioportal. These bioinformatic data will provide insight on premetastatic lung transcriptome, and potentially reveal therapeutic targets against breast cancer progression.

Dr. Mikella Robinson (postdoctoral fellow), SDSU - Biology

Characterization of SOX2, OCT4, and Nanog in Ovarian Cancer Tumor-Initiating Cells

High Grade Serous Ovarian Cancer (HGSOC) is the most lethal gynecological disease, with 70% of advanced disease relapsing within 18 months. Recent studies suggest that tumor-initiating cells (TICs) support chemoresistance and relapse in HGSOC. Identification of TICs has relied on surface markers such as CD133, CD44, CD117 or high activity of aldehyde dehydrogenase (ALDH), however these markers are not consistent across patients. Stem cell transcription factors Sox2, Oct4, and Nanog are promising markers because they function to support long-term self-renewal, multipotency, and quiescence. We hypothesize that Sox2, Oct4, and Nanog may more reliably identify ovarian cancer TICs. Using four HGSOC cell lines, three undefined serous lines, and one non-serous line we evaluated Sox2, Oct4, and Nanog expression, spheroid formation efficiency, proliferation, chemotherapy resistance and tumorigenicity. The HGSOC lines proliferated more slowly, were most chemoresistant, and had the highest expression of Sox2, Oct4, and Nanog. High Sox2 expression correlated with early tumorigenesis in a mouse model and with recurrent disease in clinical data sets. Overall our findings suggest that Sox2, Oct4, and Nanog correlate with TIC features and may more reliably identify TICs in HGSOC. Better identification of TICs will enhance our understanding of TIC biology to facilitate the development of therapies to overcome chemotherapy resistance and ovarian cancer relapse.

Katherine Jensen (graduate student), Chico State - Nutrition/Food Science

Effects of Sulforaphane on proliferation and invasion capacity of triple negative breast cancer cells exposed to tumor-associated macrophages

Triple negative breast cancer (TNBC) is characterized by high proliferation rates and an increased likelihood of invasion and metastasis. Tumor cells and cells located in the tumor microenvironment (TME), such as tumor associated macrophages (TAMs), interact through signals (e.g., cytokines) to promote cancer progression. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has been shown to reduce tumor growth in animal models and decrease invasion markers in TNBC cells grown in isolation. However, presence of TAMs in the TME can enhance tumor cell proliferation and invasion. The objective of this study was to determine if SFN can reduce proliferation and invasion of TNBC cells grown under the influence of TAM's. A conditioned-media approach was used to model non-contact interactions found in the TME using THP-1 human monocytes and MDA-MB-231 human TNBC cells. THP-1 cells were differentiated in 50% (v/v) cancer-conditioned media to create TAMs. MDA-MB-231 cells were grown in 50% (v/v) TAM-conditioned media for the remaining experiments. Cells were then treated with SFN (10 μM) or DMSO vehicle control. Proliferation was measured by an MTT-based colorimetric assay. Invasion capacity was measured by a Transwell Invasion assay. ANOVA and t-tests were used to determine statistical differences. Significant reduction in cell proliferation for the TAM-educated MDA-MB-231 cells after 24 hours (p=0. 0132) and 48 hours (p=0. 0190) of SFN treatment.

Roland Lacap (graduate student), CSU Northridge - Biology

The Roles of CXCR4 and CXCR7 in Melanocyte and Melanoma Motility

Chemokines are signaling proteins released by cells in response to chemical stimuli in their environment. The chemokine stromal derived factor 1 (SDF1) has been regularly studied due to its role in the growth and metastasis of multiple cancers, including melanoma. SDF1 has two known receptors: CXCR4 and CXCR7. Previous research has focused on CXCR4 receptor signaling, which influences many cell responses, among them the migration of neural crest cells and the amount of receptor expression upregulation in melanoma. CXCR7 receptor signaling has been shown to influence melanocyte migration and constrain melanoma tumor growth in vivo, while CXCR4 receptor signaling could not. It is not known, however, how relevant CXCR7 may be in the capability of melanocyte and melanoma migration. Here, the potential roles of CXCR4 and CXCR7 receptor signaling was studied in melanocyte and melanoma migration in vitro by genetically silencing both receptors. The results suggest that the CXCR7 receptor is more important than the CXCR4 receptor for the migratory capabilities of melanocytes and melanoma cells. These findings suggest that down regulating or blocking the CXCR7 receptor through targeted therapies may show a substantial effect in melanoma treatment.

Jane Odango (graduate student), CSU Northridge - Chemistry/Biochemistry

The Effects of Varying DNA flap length from 20 to 50 Deoxynucleotides on the Mechanism of Single-Strand Annealing in Saccharomyces cerevisiae

Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double-strand breaks (DSBs). This project focuses on a repair pathway known as single-strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3 overhanging single-stranded DNA “flaps” which are cleaved by the Rad1-Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1-dependent recruitment of Rad1-Rad10 to damage sites and SSA repair proficiency are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring Saw1 for optimal SSA repair in vivo. In order to investigate the importance of DNA flap length on Rad1-Rad10 and Saw1 recruitment, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB-RFP), allowing us to monitor the recruitment of Rad10-YFP via fluorescence microscopy. The percentages of cells containing colocalized foci in strains either wild-type or mutant in Saw1 were quantified, revealing that Saw1-mediated recruitment of the Rad1-Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to the DSB site across all flap length substrates via experiments with a Saw1-CFP strain. Current efforts are underway to determine the Saw1-dependence of proficient SSA repair.

Jonathan Hakimian (undergraduate student), CSU Northridge - Chemistry/Biochemistry

DNA Double-Strand Break Repair; Examining the Role of SLX4 in Checkpoint Signal Dampening in Dividing Cells

The study of DNA repair pathways and their interaction with DNA damage plays a pivotal role in understanding the mechanisms of cancer, a disease that has become an increasing concern for humanity. Our work in the lab focuses on the function of Slx4, a protein participating in checkpoint signal dampening, which governs two DNA Double-Strand Break (DSB) Repair pathways: Single-Strand Annealing (SSA) and Synthesis-Dependent Strand Annealing (SDSA). We have evidence that the Rad1-Rad10 endonuclease complex, which is required in SSA and SDSA, partially requires Slx4 for its recruitment to DSB sites in S phase. We hypothesize that checkpoint signal dampening by Slx4, which also occurs in S phase, results in this SLX4-dependent recruitment of Rad1-Rad10 to DSBs. By constructing Slx4 phosphorylation-mutant S. cerevisiae strains (baker’s yeast) in which Slx4 is defective in checkpoint signal dampening, we can analyze the recruitment of Rad1-Rad10 to DSBs in absence of this function. Preliminary trials were successful in optimizing cell cycle arrest/release experiments, in which we analyzed cell cycle-synchronized cultures by fluorescence microscopy and flow cytometry. We plan to use these strains and optimized experimental conditions to determine if Slx4 checkpoint signal dampening function promotes SSA in DSB repair in S phase. A better understanding of the biochemical underpinnings of DSB repair may aid in the design and development of a more effective means of cancer treatment.

Dr. Matthew Jones (postdoctoral fellow), University of Manchester - Biology

Cell-Matrix Adhesion and the Cell-Cycle

Precise temporal and spatial regulation of cell cycle progression is required for both metazoan development and maintenance of adult tissue. Cell adhesion to the extracellular matrix (ECM), which is mediated by the assembly of integrin-dependent adhesion complexes (ACs), is required for cell survival and progression through the cell cycle. The ability of cancer cells to evade this requirement has long been a hallmark of tumour development. Furthermore, changes in ECM stiffness and composition that alter ACs contribute to cancer progression and metastasis. Rounding up of cells occurs during mitosis which requires the coordinated disassembly of ACs, before they are reestablished following cytokinesis. Failure of the inter-dependence of adhesion and cell cycle progression results in aberrant cell division, contributing to the progression of a number of diseases. At present, it is largely unclear how the composition of ACs changes during cell cycle progression and how these changes contribute to accurate cell division. I have identified robust compositional changes in ACs as cells progress through the cell cycle, from an initial increase in ACs in S phase, to disassembly of ACs in G2 prior to mitosis. The disassembly of ACs in G2 requires CDK1 inactivation via Wee1 phosphorylation and is required for efficient mitosis to occur. Thus, I have identified a novel mechanism linking the cell cycle machinery to regulation of ACs and the cytoskeleton via the modulation of CDK1 activity.

Ivan Salladay-Perez (graduate student), CSU Los Angeles - Biology

Assessing the Impact of an Adenylate Kinase Isoenzyme on AMPK and mTOR Signaling Pathway through Metabolic Profiling in both Brain and Lung Cancer Cell Lines

In a variety of human cancers, it is well observed that altered bioenergetics must be sustained in order to maintain cancer cell virulence. Recent events show that without the expression of an AK4, a mitochondrial matrix enzyme. We demonstrate that in response to perturbed AK4 expression in cancer cells, two critical homeostatic signaling pathways, the AMPK and a mTOR pathways, are significantly altered. Both mTOR and AMPK proteins govern a metabolic pathway in which they sense cellular energy levels and nutrient status in response to regulate cancer cell growth and/or proliferation. AK4 also has an impact on other bioenergetic pathways. For instance, it’s been observed that AK4 can guanine nucleotide metabolism and sustain critical energy flux of nucleotides in the cell. Additionally, results show altered adenosine nucleotide pool concentrations in response to loss of AK4 expression. Because AK4 expression appears to regulate cellular metabolic responses at multiple levels in cancer cells, further analyses by obtaining a metabolomic profile is necessary to highlight the global impact AK4 exerts on all the metabolites in the cancer cell system. Cell lines will be engineered without the AK4 gene in glioblastomas, lung adenocarcinomas, and HeLa cells. It is hypothesized that without the expression of an AK4 protein there will be disruptions in global nucleotide pools. The outcome of this study will provide a better understanding of how AK4 impacts cancer cell metabolism.

Hafridha Hadi (graduate student), CSU Northridge - Chemistry/Biochemistry

Construction of a Specialized Yeast Strain containing YEN1-yEMRFP to Study Holliday Junction Resolution

Double-strand breaks in DNA can be harmful if left unrepaired. Eukaryotes use repair mechanisms, some of which involve intact chromosomes that template repair via an intertwined intermediate known as a Holliday Junction (HJ). In S. cerevisiae, the Yen1 endonuclease resolves HJs but it’s unclear whether it localizes to anaphase bridges, the final intertwined regions during chromosomal separation. Yen1 is present in lower abundance relative to other proteins, therefore, microscopy experiments with fluorescently labeled Yen1 suffers from low signal-to-noise. Our aim is to clone a yeast strain with a bright yeast Enhanced Monomeric Red Fluorescent Protein (yEMRFP) under the strong galactose promoter, thereby allowing us to track the position and timing of Yen1 during HJ resolution. To construct this strain, we used adaptamer-mediated Polymerase Chain Reaction, DNA digestions, and ligations to prepare two DNA plasmids. Each plasmid contains partial, but overlapping fragments of the URA3 auxotrophic selection marker gene, one copy of the yEMRFP gene, and flanking sequences of the target yeast chromosome to direct integration to the N-terminus of YEN1. Both plasmids were transformed into yeast and screened by PCR and DNA sequencing, which showed that the genetic elements from the plasmids were successfully integrated into the yeast genome. This yeast strain will produce an improved signal-to-noise in fluorescence microscopy work to elucidate the role of Yen1 at anaphase bridges.

Leticia Reque (graduate student), CSU Northridge - Chemistry/Biochemistry

Investigating the Members of the Aldo-Keto Reductase Family of Enzymes as an Adaptive Mechanism to Glyoxalase 1 Inhibition

Breast cancer and prostate cancer are the two of the most frequently 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 inhibition of Glo 1 by the glutathione analogue, BBGC, results in cell death in a panel of breast, prostate, and ovarian cancer cell lines. We hypothesize that Glo 1 inhibition leads to the accumulation of reactive carbonyl species and that AKRs are uniquely equipped to act as a compensatory detoxification system. Members of the aldo-keto reductase (AKR) superfamily are up-regulated in cancer. AKR1C3 functions in the biosynthesis of steroid hormones such as testosterone and dihydrotestosterone, the potent androgen receptor ligands. We subjected a panel of breast and prostate cancer cell lines to treatment with EC50 concentrations of the Glo 1 inhibitor, BBGC. 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 are establishing a method for absolute quantification using standards of known concentrations and qRT-PCR to obtain cycle threshold values to produce a standard curve. This suggests the potential for pairing Glo 1 inhibitors with inhibitors of specific members of the AKR super-family in certain tumor types.

Matthew Wallace (graduate student), CSU Northridge - Biology

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. 4553 and 1083 proteins were identified in WCEs and IACs, respectively. Of these, 199 and 62 proteins changed upon ITGA1 knockdown in WCEs and IACs, respectively. ITGA1-induced IAC components that remained unchanged in WCEs upon ITGA1 knockdown were further analyzed by Cytoscape to generate a network interactome and grouped by clustering coefficient and connectivity. Notably, the interactome converged on ITGA1-induced IAC components KIFC1 and PLK1. While previous studies have implicated PLK1 in PDAC therapy resistance, KIFC function in this disease remains unknown. Analysis of PDAC patient survival in relation to expression levels of ITGA1, KIFC and PLK1 revealed significantly reduced relapse-free survival when both ITGA1 and PLK1 were elevated. These data suggest that ITGA1 may recruit PLK1 into PDAC adhesion complexes to drive disease progression and ongoing work aims to discern the role of ITGA1-PLK1 signaling in focal adhesion turnover and PDAC cell migration.

5-minute presentation Format

Joanna Maddela (undergraduate student), CSU Northridge - Biology

Regulation of eIF5A1/2 Subcellular Localization in Breast Cancer

Breast cancer has an overall five-year survival prognosis of <27%, being the leading cause of cancer-related deaths in 2020 for women in the United States. By pharmacologically inhibiting deoxyhypusine synthase (DHPS)-mediated eIF5A1/2 hypusination in human breast cancer cell lines using GC7, we were able to demonstrate the inhibition of PEAK1 translation, cell viability and PEAK1-mediated TGFβ/fibronectin-driven breast cancer metastasis. In the past, eIF5A1/2 has been linked to PEAK1 protein translation and its tumorigenic activities to post-translational activation of the eIF5A1/2. Following these results, we began to analyze and observe the sub-cellular localization of eIF5A1/2 in breast cancer cells upon changing serum percentage in the media, stimulation of TGFβ/fibronectin, and across different cell lines. In addition, we would also be able to indirectly observe PEAK1 protein expression. We hypothesize that there is a correlation between eIF5A1/2 sub-cellular localization and the effects of TGFβ/fibronectin in the PEAK1 pathway. We tested our hypothesis by performing immunofluorescence assay and scoring cells based on eIF5A1/2 staining. Ongoing work aims to quantify eIF5A1/2 nuclei cytoplasm intensity ration in hopes to show intracellular heterogeneity in human breast cancer cells. This work has the potential to identify efficacious combinatorial targeted approaches to inhibit breast cancer progression.

Siyuan Li (undergraduate student), CSU Northridge - Engineering/Bioengineering

Printability Evaluation of 3D Bioprinted MIA PaCa-2 Human Pancreatic Cancer Model

3D Bioprinting executes the idea of utilizing printed biocompatible tissues and organs for the study of cell behaviors. This study’s objectives include: a) bio-print a MIA PaCa-2 human pancreatic cancer cell model surrounded by stromal cells; b) evaluate the efficacy of the tissue models produced by the microextrusion 3D bioprinting technique relative to the growth and development of stromal cells. In this study, a custom microextrusion 3D bioprinter was built to print the malignant tissue constructs to study the motility of malignant and stromal cells. It includes three pneumatic syringe printing heads and utilizes three bioinks: two cell-laden alginate hydrogels and one sacrificial material. The cell-laden hydrogels contained hyaluronic acid (HA), glycerol, gelatin, sodium alginate, and stromal cells or cancer cells. The sacrificial material included a 10% w/v glycerol solution and pluronic f-127. To find the most effective hydrogel, different formulas under different conditions along with their effects were investigated. The following variables were tested: three different ratios of low and high molecular weight sodium alginate; nine different total alginate concentrations; four different incubation times; and the effect of calcium ions on the hydrogel. After comparing each hydrogel’s printability, viscosity, and post-print swelling ratio, the most effective hydrogel preparation procedure was determined.

Charles Metzler-Winslow (undergraduate student), CSU Los Angeles - Physics

Toward Accurate Short-Range Physically-Derived Models for Charge in Molecular Dynamics Simulations and Downstream Importance in Rational Drug Design

A smeared-charge model is presented pursuant to the inability of partial point charge models to capture short-range screening effects; the importance in molecules with deep potential wells among numerous molecules for which rough dynamical intuition fails is discussed. The principal relevance to rational drug design is also discussed.

Jennifer Anderson (undergraduate student), CSU Northridge - Chemistry/Biochemistry

Investigating the Role of NRF2 and DJ-1 in Response to Glyoxalase 1 Inhibition in Breast, Prostate and Ovarian Cancer Cells

The accumulation of reactive oxygen species (ROS) and reactive carbonyl species (RCS) without proper removal by detoxification systems can increase one’s susceptibility to neurodegenerative diseases, aging, and cancer. The Keap1-Nrf2 system plays a critical role in sensing ROS and RCS and expressing genes involved in cellular defense against these insults via the Nrf2 transcription factor. The binding of Nrf2 to the antioxidant response element (ARE) leads to the expression of members of the aldo-keto reductase superfamily and genes involved in glutathione biosynthesis. Separately, DJ-1/PARK7 is up-regulated in a variety of tumors and has recently been found to have deglycase activity. This activity is purported to be involved in the removal of reactive carbonyl species adducts from protein and DNA. We are investigating the relationship between DJ-1, Nrf2, and the Glyoxalase 1 (Glo1) detoxification pathway in prostate, breast, and ovarian cancer. We used the glutathione analogue, BBGC, to inhibit Glo 1, which is over-expressed in a wide variety of tumors and cancer cell lines and have analyzed Nrf2 nuclear translocation and DJ-1 expression via western blot and AKR expression levels via quantitative real-time PCR (qRT-PCR). Taken together, the robust levels of DJ-1 and Nrf2 translocation do not appear sufficient to surmount the elevated RCS generated by Glo 1 inhibition in cancer cells.

Esther Alarcon (undergraduate student), CSU Los Angeles - Chemistry/Biochemistry

Exploring Chemo-Resistant DARPP-32/t-DARPP's Mega Complexes Through Gel Filtration Chromatography

The PPP1R1B gene is frequently overexpressed in breast cancer cells with acquired resistance to trastuzumab (Herceptin). Two protein products were characterized: Darpp-32, consisting of 208 amino acids and t-Darpp, a truncated version of Darpp-32 consisting of 168 amino acids. t-Darpp is associated with trastuzumab resistance. Overexpression of t-Darpp leads to increased protein kinase A (PKA) activity and phosphorylation of AKT1 (alias protein kinase B [PKB]) leading to apoptotic evasion and cell proliferation. Recombinant t-Darpp (19kDa) displays a surprisingly large Stokes radius of 4.4nm, likely due to the fact that this protein has unstructured regions. Chemical crosslinking studies conducted on cultured breast cancer cells indicate that t-Darpp forms high molecular weight complexes well over 150 kDa. Gel filtration chromatography of soluble extracts from breast cancer cells demonstrates that human t-Darpp/Darpp-32 form two distinct megacomplexes. Both megacomplexes eluted outside the linear resolving range of the column. One megacomplex is well over 600kDa and the other megacomplex is closer to 600kDa. Copurification of t-Darpp/Darpp-32 followed by mass spectrometry identified candidate proteins that copurified with t-Darpp: IPTKB, Sp110, P4HB, calreticulin, EVL, PDIA4, MyoGEF, ERp72 and secerning-3. This suggests that t-Darpp/Darpp-32 forms high molecular complexes in the HER2+ breast cancer patients which may be critical for its trastuzumab-resistance properties.