Dr. Cory Brooks (Faculty), CSU Fresno - Chemistry &/or Biochemistry
Role of Mucin glycosylation in the binding of a therapeutic antibodyIn cancer cells, the mucin protein MUC1 is overexpressed and undergoes truncated glycosylation that exposes cryptic peptide sequences. Since these sequences are cancer specific, they represent a target for therapeutic antibodies. We investigated the truncated glycosylation on binding by the therapeutic antibody AR20.5. We explored the affinity of AR20.5 to a synthetic cancer specific MUC1 glycopeptide and peptide. The antibody bound the glycopeptide with an order of magnitude stronger affinity than the peptide. Given these results we hypothesized that AR20.5 must specifically bind the carbohydrate as well as the peptide. Using X-ray crystallography we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific polar contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of binding contacts supports a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide that is recognized by the antibody. High affinity binding of AR20.5 to the MUC1 glycopeptide is not driven by specific antibody-antigen contacts, but rather alters the conformational equilibrium of the antigen. This study demonstrates a novel mechanism of antibody-antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.
Dr. Kevin Corbett (Faculty), UCSD - Cell and Molecular Medicine
An uphill battle: TRIP13 maintains genome integrity by mediating both activation and inactivation of the mitotic spindle assembly checkpointThe mitotic spindle assembly checkpoint (SAC) ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), an inhibitor of the Anaphase Promoting Complex/Cyclosome (APC/C), at unattached kinetochores in prometaphase. The AAA+ ATPase TRIP13 is a key checkpoint mediator, but its molecular mechanisms and biological roles have remained largely mysterious. TRIP13 is overexpressed in most cancers, yet its deletion also causes high susceptibility to certain cancer types, suggesting an unappreciated complexity in TRIP13's contributions to genome integrity. Here, we show biochemically that TRIP13, with the adapter protein p13comet, can catalyze MCC disassembly by partially unfolding its MAD2 subunit. Next, using a system to inducibly degrade TRIP13 in live cells, we show that acute loss of TRIP13 delays checkpoint silencing due to an inability to disassemble the MCC. Paradoxically, we also find that long-term depletion of TRIP13 also eliminates SAC activation. This unexpected effect is due to a spontaneous conformational change in free MAD2 in the absence of TRIP13, which prevents its incorporation into the MCC. Together, our data reveal that TRIP13 plays two opposing roles in the SAC through a single biochemical mechanism: (1) converting free â€œclosedâ€ MAD2 to the â€state to support MCC assembly and checkpoint activation, and (2) actively disassembling MCC through MAD2 conformational conversion to promote checkpoint silencing.
Dr. Eric Kelson (Faculty), CSU Northridge - Chemistry
Anti-tumor activity of DNA-binding ruthenium terpyridine complexesTpys (2,2’:6’,2”-Terpyridines) and polypyridine ruthenium complexes have been reported to be anti-tumor agents that exhibit promising selectivity for tumors likely due to their active transport into cancer cells. The Kelson group has prepared Tpy based ruthenium complexes bearing DNA intercalating groups as new anti-tumor candidates designed to exploit active transport. Specifically, four complexes have been investigated in detail: (Tpy)Ru(TpyTpy)(2+) (Complex 1) [where TpyTpy=4’,4”"-bis(2,2’:6’,2”-terpyridine), (AnthTpy)Ru(Bpy)Cl(+) (Complex 2) [where AnthTpy=4’-(9-phenanthryl)-2,2’:6’,2”-terpyridine and Bpy=2,2’-bipyridine], (AnthTpy)RuCl2(DMSO) (Complex 3) (where DMSO=dimethylsulfoxide), and (Tpy)Ru(Dppz)Cl(+)(Complex 4) (where Dppz= dipyrido[3,2-a:2′,3′-c]phenazine). All four complexes bind DNA strongly (with the last three by intercalation) and exhibit significant toxicity (with values of EC50 from 30 to 6 µM) toward several tumor lines (MDA-MB-231, MDA-MB-468, MCF-7, and HepG2). In tests with active transport inhibitors, the toxicity of Complex 1 was attenuated by added spermine suggesting assisted entry into tumor cells via polyamine transporters. This is consistent with the repetition of nitrogen donors within the pendent Tpy group chemically resembling natural polyamines. The results from complex 1 have inspired a second generation of DNA intercalators bearing other polyamine-like groups for cell entry. Preliminary results with these compounds will be presented.
Dr. Byron Purse (Faculty), San Diego State - Chemistry &/or Biochemistry
Fluorescent Nucleoside Analogues for Nucleic Acid BiophysicsThe goal of our research is to develop new fluorescent modifications of DNA and RNA that minimally perturb structure but provide novel capabilities for biophysical studies on the copying, maintenance, and expression of the genetic code. Errors in these processes are the fundamental drives of cancer. Towards this aim, we designed and synthesized a series of 11 new fluorescent, tricyclic cytidine analogues with systematic placements of electron donating and withdrawing groups and studied their fluorescence properties in nucleic acids. Among these compounds, we discovered that the new analogue 8-DEA-tC offers the most powerful known fluorescence turn-on response to matched base pairing in duplex DNA and RNA, promising applications in monitoring DNA/RNA secondary structure and for detecting single nucleotide polymorphisms. Another compound, 8-Cl-tCO, exhibits vibrational fine structure in its fluorescence spectrum that is responsive to base pairing and stacking and offers the potential for a new way to monitor the dynamics of nucleic acid structure. Our collected results reveal patterns relating the design of nucleobase analogues to their fluorescent responses to base pairing and stacking. Current efforts including developing applications of these new fluorescent probes to nucleic acid biophysics, and we are seeking collaborators interested in applying the probes to the molecular biology of cancer.
Dr. Mariano Loza-Coll (Faculty), CSU Northridge - Biology
Heterochromatin Protein 1 (HP1) inhibits the tumorigenic overproliferation of stem cells induced by the ectopic activation of the Jak/STAT pathway in the Drosophila testisRecent work has explored the role of heterochromatin and heterochromatin-associated proteins in the regulation of stem cell homeostasis. However, less is known about their roles in overproliferative stem cells. Here we used ectopic activation of the Jak/STAT pathway in germ and somatic stem cells of the D. melanogaster testis to force the tumorigenic overproliferation of two different stem cell populations in vivo, and thus probe the function of Heterochromatin Protein 1 (HP1) in overproliferative stem cells. Overexpression of HP1 in either cell type suppressed the tumorigenic overgrowth of testes caused by ectopic Jak/STAT activation. Interestingly, HP1â€™s growth suppressive effect correlated with different phenotypes, depending on whether HP1 was overexpressed in somatic or germ cells. Furthermore, our data suggest that both cell-autonomous and non-autonomous mechanisms may underlie the proliferative suppression triggered by HP1. Our results support the use of this model of tumorigenesis in the Drosophila testis to not only better understand the regulation of tumorigenic stem cell overproliferation by heterochromatin in vivo, but also to explore other cross-regulatory mechanisms between different populations of overproliferative stem cells.
Dr. Thomas Minehan (Faculty), CSU Northridge - Chemistry
Shape-selective DNA-binding small molecules for targeting the CYP19 genetic polymorphism (TTTA)n present in patients with increased risk of breast and prostate cancersCell permeable small molecules that bind the major groove of DNA have the potential to regulate gene expression either through the disruption of transcription factor-DNA complexes in gene promoters or by interfering with the action of RNA polymerase in the coding region of genes. Utilizing the tools of molecular modeling and synthetic organic chemistry, we have designed and prepared a series of sterically bulky dimeric and trimeric derivatives of the histochemical stain crystal violet which show a preference for binding non-alternating AT tracts of B*-form DNA; such sequences are known to contain a wider major groove relative to canonical B-form DNA. Biophysical studies employing fluorescence and circular dichroism spectroscopy confirm that these cationic molecules occupy the major groove of DNA and make strong electrostatic contacts with the phosphate backbone. Electrophoretic shift analysis suggests that these molecules are also capable of inhibiting the association of DNA-binding proteins with their cognate sequences in vitro. These molecules or their derivatives may selectively accumulate in genomic regions containing (TTTA)n polymorphisms, which are structurally similar to non-alterating AT tracts; such polymorphisms in the CYP19 (aromatase) gene have been reported to be associated with an increased risk of breast and prostate cancer. Shape-selective DNA-binding molecules can be used to clarify the biologic function of the CYP19 polymorphism.
Dr. Nathan Lanning (Faculty), CSU Los Angeles - Biology
Mitochondrial AK4 expression coordinates dual AMPK and mTOR activationTwo opposing signaling pathways which are critical regulators of cell growth and metabolism and which are also commonly mis-regulated in cancer are the AMPK and mTOR pathways. mTOR generally promotes cellular growth and proliferation, while AMPK inhibits mTOR and generally suppresses growth and proliferation. Both pathways are acutely responsive to cellular nutrient and energy levels and demands. We have identified the expression of the mitochondrial adenylate kinase 4 (AK4) to regulate cellular adenosine levels and concurrent activation of AMPK and mTOR. Each of these pathways are important for maintaining cellular homeostasis, and dysregulated mTOR and AMPK signaling are both implicated in numerous cancers. We have found AK4-expression dependent activation of AMPK and mTOR to enhance proliferation and activate other cellular responses such as autophagy. We have additionally found that altered AK4 expression enhances the ability of cancer cells to survive under high ROS conditions by enhancing redox homeostasis. These findings position AK4 upstream of both signaling pathways, with AK4 expression able to coordinate activation of both pathways and provide cells with the ability to survive under stresses associated with oncogenic transformation while at the same time initiating pro-proliferative signaling.
Dr. PaulChris Okpala (Faculty), CSU San Bernardino - Health Science & Human Ecology
Social Support Provision for the Cancer Patients and their Associated Socioeconomic EffectsLittle has been done to assess the effects of social support provided to the cancer patients. This study assesses how the provision of social support to the cancer patients affects their health and socioeconomic status. A quantitative study design approach was used, which involved an evaluation of a total of forty-three existing articles. Collected data were analyzed using ANOVA, regression, and correlation statistics. Logistic regression showed that mental health disorders among cancer patients were significantly reduced among those that were involved in the provision of social support (F=1.344, P=0.022). The cancer patients that were involved in the provision of social support also reported enhanced relationship within their families. However, the provision of social support was shown to be associated with a significant reduction in financial stability among these cancer patients (P=0.001), which was observed to vary based on ethnic background. This study recommends the adoption of social and financial support as a means of enhancing the health of the cancer patients.
Dr. Chen Huai-Lu (Application Scientist), NanoCellect Biomedical Inc. - Biology
WOLF Cell Sorter platform for single cell isolationThe heterogeneity among individual cells play critical roles in normal development, physiology, diseases formation, and the responses to treatments. Currently, single cell isolation methods include using traditional fluorescent activated cell sorting or laser-assisted microdissection in the core lab, micromanipulation by microinjector, or utilizing limiting dilution in the individual lab. However, an easy and efficient system for single cell isolation is still lacking. Here we present the use of a novel flow cytometry system, the WOLF Cell Sorter, for rapid and reproducible single cell isolation dispensed into 96 well or 384 well culture plates, as well as on 96 well PCR plates. One of the features is higher viability due to lower shear stress. To evaluate this feature, we enumerated the cell number in each well, cultured the plates, and monitored the cell growth from day one through day 14. The cell outgrowth rate results showed more than 70% of the single cells formed colonies successfully under tested conditions. The initiation time for setting up the instrument takes approximately 15 min and 8 min to dispense into each 96 well culture plate. The results reveal that the WOLF cell sorting platform can efficiently isolate targeted single cells while maintaining high viability and can be used for the isolation of CRISPR modified stem cells, T-cells, antibody production CHO cells, and for downstream single-cell genomic analysis.
Dr. Farhana Runa (Post-doc), CSU Northridge - Biology
Functional studies of RAI14 during microenvironment remodeling and progression in pancreatic cancerThe extensive desmoplastic response in the pancreatic ductal adenocarcinoma (PDAC) microenvironment is one of the primary hurdles toward developing effective therapies to combat this deadly malignancy. Since O-GlcNAc modification of SEC23A mediates proper trafficking of collagen (a primary constituent of the PDAC micoenvironment), we reasoned that SEC23A interacting partners which are upregulated in PDAC may yield novel strategies for targeting the PDAC stroma. In this regard, we identified RAI14 (Retinoic Acid Induced 14 or Ankycorbin or NORPEG) as a candidate SEC23A binding partner using GlcNDAz-mediated crosslinking and mass spectrometry, and ongoing work aims to confirm/validate these preliminary biochemical results. Notably, both RAI14 and SEC23A are among the top 6% of genes that are significantly upregulated in pancreatic cancer tissue relative to normal pancreatic tissue. Further, we report that RAI14 co-localizes with actin stress fibers in PDAC cells and that RAI14 knockdown reduces PDAC cell migration and proliferation/survival. Using the CRISPR-CAS9 system, RAI14 was depleted from PDAC as well as chondrosarcoma and non-malignant kidney epithelial cells to further test for collagen expression/secretion, chemotherapy responsiveness and tumor growth/metastasis. The long-term goal of these studies is to elucidate the mechanistic basis for PDAC progression and therapy resistance.
Dr. Robert Guth (Post-doc), CSU Northridge - Biology
Stromal-to-Epithelial PEAK1-Dependent Signaling Drives Tumor Growth and Therapy Resistance in Breast Cancer by Enriching for PI3K/NFkB-Expressing Stem-Like CellsDevelopment of breast cancer therapies is impeded by the extensive cellular and molecular heterogeneity in this disease. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is a cytoskeleton-associated kinase that promotes tumor progression and mesenchymal cell states. PEAK1 was recently found to also be upregulated in the stroma of breast tumors, and this expression predicts breast cancer recurrence and is associated with disease relapse. We report that patient-derived breast cancer-associated fibroblasts and mesenchymal stroma cells (MSCs) express PEAK1 and that co-xenografting of these cells with HER2- or ER-positive breast cancer cells (BCCs) promotes primary tumor growth and targeted therapy resistance in vivo. Notably, PEAK1 knockdown in MSCs abrogated these responses, as well as protective effects on HER2-positive BCCs in vitro. Multiplex immunofluorescence (cycIF) studies at single-cell resolution revealed that PEAK1 expression in MSCs is required for survival of SOX2-positive BCCs that remain high in Akt and p65-NFï«B signaling following lapatinib treatment. Protein array screening of PEAK1 knockdown MSCs identified depletion of soluble factors with known protumorigenic functions. Analyses of clinical databases support targeting of these factors to sensitize breast cancer cells to targeted therapies, thus suggesting new strategies for improving patient outcome.
Ranelle Buck, CSU Northridge - Biology
Expression of Wild Type and Chimeric E. coli Cytotoxic Necrotizing Factor 1 by Tumor-Targeted SalmonellaConventional cancer therapies have negative side effects, and some cancers have high rates of recurrence.Â An alternative to these therapies involves the application of tumor-targetedÂ Salmonella, which preferentially locate and grow within tumors.Â S. entericaÂ serotype Typhimurium VNP20009 is an attenuated strain that localized in tumors in human clinical trials, but did not inhibit tumor growth. One way of increasing tumoricidal activity of this strain isÂ toÂ engineer it to express a toxin. Cytotoxic necrotizing factor 1 (CNF1) is a toxin that activates Rho, Rac, and Cdc42 GTPases in eukaryotic cells, irreversibly inhibiting cytokinesis.Â In this study, the binding region of CNF1 was altered from the wild type to TGFÎ±, a member of the EGF family of proteins. As TGFÎ± is overexpressed in some tumors, it is a potential target for conferring tumor cell specificity to CNF1. This study also explored exchanging the secretion region of CNF1 with the OmpA secretion signal in an attempt to facilitate secretion of proteins in higher amounts. Although secretion of both chimeric forms was shown using a polyclonal antibody, neither of these proteins were cytotoxic toward tumor cells. As an alternative method of conferring tumor-specificity, we will explore the introduction of a hypoxic promoter to take advantage of the conditions commonly found within tumors. This project has the potential to yield a targeted treatment and functional delivery system suitable for studies in animal models.
Samantha Hain, CSU Northridge - Biology
The Roles of CXCR4/7 in Melanocyte and Melanoma MotilityChemokines are signaling proteins released by cells in response to chemical stimuli. 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 protein receptors: CXCR4 and CXCR7. Previous research has studied only the CXCR4 receptor, which influences migration of neural crest cells and is upregulated in melanoma. Once neural crest cells differentiate into melanocytes CXCR7 influences their migration and has been found to constrain melanoma tumor growth in vivo. My preliminary data has shown that melanoma cells do not migrate through normal contact inhibition of locomotion. Cell to cell contact causes an inhibition of migration in normal cells, whereas melanoma has been seen to lack this regulatory mechanism in regard to locomotion. Therefore, I anticipate downregulation or inhibition of the CXCR7 receptor will be more influential than the CXCR4 receptor in the development of a malignant phenotype in melanocytes. Furthermore, I plan to narrow down the specific domain of the CXCR7 receptor that turns the melanocytes into melanoma.
Fred Fregoso, CSU Northridge - Chemistry/Biochemistry
The necessity of Saw1 in recruiting Rad1-Rad10 to single-strand annealing sites increases as a function of increasing DNA flap length in S. cerevisiaeAccumulation of DNA double-strand breaks (DSBs) has cytotoxic consequences including genomic instability and cell death. When a DSB occurs between two DNA repeats, cells may employ one of the HR modes known as single-strand annealing (SSA), a non-conservative repair pathway linked to genome rearrangements and oncogenesis. In the yeast S. cerevisiae, an SSA intermediate bearing 3â€² overhanging single-stranded DNA flaps are cleaved by the protein complex Saw1-Rad1-Rad10. In vivo studies show that Rad1-Rad10 recruitment to long flaps (~500 bases) is Saw1 dependent, however not when flaps are short (~10 bases). To determine the flap length triggering a need for Saw1, specialized fluorescent yeast strains were constructed containing a labeled RAD10 gene (Rad10-YFP) and an inducible DSB site tagged by TetR-RFP protein (DSB-RFP). The DSB site is flanked by DNA repeats separated by varying distances, which produce flap intermediates during SSA of either 20, 30, or 50 bases. We monitored the recruitment of Rad10-YFP to DSB-RFP sites via fluorescence microscopy and compared results from strains that were either wild-type in SAW1 or deleted of SAW1 to determine whether Rad10-YFP recruitment is affected by the absence of SAW1. Results show a trend confirming that the requirement for Saw1 is a function of flap length, and indicating that flaps of ~20 bases trigger a partial requirement for Saw1 that becomes more pronounced in 30-base flaps and absolute in 50-base flaps.
Vander Karamjot, CSU Fresno - Chemistry/Biochemistry
An evaluation of Palladin overexpression in pancreatic cancer cellsPancreatic cancer (PC) is the fourth leading cause of cancer in United States with overall survival rate of less than 5% (ACS, 2018). Its aggressive nature and late onset of physical symptoms lead to poor prognoses. Recognition of biomarkers is clearly an imperative undertaking for the disease. To tackle this problem, we evaluated the secreted protein profile of pancreatic cancer cell lines derived from different stages. In total, nine human pancreatic cancer cell lines were successfully adapted into serum-free environments from which conditioned media was collected by a combination of ultracentrifugation, molecular weight cutoff, protein precipitation, gel-based separation, and tryptic digestion followed by MS protein identification. A small subset of proteins has been identified and validated biochemically including Palladin (PALLD), a pancreatic cancer susceptibility protein shown to be up-regulated in previous studies. PALLD is a scaffolding protein that supports remodeling of the actin cytoskeleton in conjunction with integrins at focal adhesions to coordinate cell migration. We demonstrate a plausible interaction between an isoform of PALLD and integrin (ITGB5) indicative of the increased motile phenotype in PC. Taken together, our results suggest that PALLD overexpression is associated with enhanced motility in pancreatic cancer cells and could represent a potential biomarker or therapeutic strategy against pancreatic cancer progression.
Carlos Gonzalez, CSU Los Angeles - Biology
AK4 regulates cellular bioenergetics and redox bufferingIn order to maintain cellular homeostasis, mitochondria integrate bioenergetic, metabolic, and redox cues. Signals both converge on and emanate from mitochondria to respond to varied cellular stressors. Therefore, mitochondrial proteins must play critical roles in maintaining cellular homeostasis. In a search for mitochondrial proteins that impact cellular bioenergetics, we identified the adenylate kinase, AK4, as the most potent regulator of ATP levels out of >1,000 mitochondrial proteins. We also found AK4 expression to significantly impact cells' ability to respond to energetic stressors by maintaining NADP(H) pools, regulating cellular redox buffering, and engaging autophagy. Together, these effects of suppressed AK4 expression allow cells to thrive in the context of nutrient deprivation-induced stress. The profound effects of AK4 expression on bioenergetics and survival under stress position AK4 as a potential master regulator of mitochondria-dependent cellular homeostasis.
Macias Alejandro, CSU Northridge - Biology
Examination of the Apoptotic role of E4BP4 in breast cancer cellsGlucocorticoids (GCs) such as Dexamethasone (Dex) are anti-cancer agents that induce apoptosis (programmed cell death) and counter side effects of chemotherapy through regulation of pro- and anti-apoptotic genes. E4BP4, a transcriptional regulator, is upregulated by GCs and is known to mediate Dex-evoked apoptosis in leukemia cells, but has been reported to promote cell survival in other models of cancer, including breast cancer. The phosphorylation state of E4BP4 and its crosstalk with the FOXO3A and AKT pathways modulate its pro-survival or pro-apoptosis functions. We hypothesize that Dex-evoked upregulation of E4BP4 and its subsequent modulation of proliferative signals may contribute to pro-survival effects of Dex in breast cancer. In the present study we are investigating the response of breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (triple negative) to Dex and Dauno (single agent and in combination) with respect to cell viability, apoptosis and regulation of pro- and anti-apoptotic genes including E4BP4, FOXO3A, BIM and BIRC3. Our data suggest E4BP4 upregulations in all treatments in MDA-MB-468 cells. Single treatment and combined treatment of Dex significantly enhances FOXO3 upregulation compared to Dauno, in correlation with increased cell death. Our data suggest that Dex and Dauno may cooperatively promote apoptosis in MDA-MB-468 cells.
Kiana Requena, CSU Northridge - Biology
Gyrase-mediated Supercoiling Modulates the Expression of the Caulobacter crescentus sciP PromoterThe location of genes on the Caulobacter crescentus chromosome is thought to regulate gene expression based on the methylation states of the promoters. As the replisome copies the chromosome, it leads to major changes in the supercoiling of DNA, which we believe controls transcription of some cell-cycle regulated genes. We treated C. crescentus cultures with the antibiotic Coumermycin A1, a DNA gyrase inhibitor. As reported for other bacteria, high levels of the antibiotic inhibited growth, whereas low levels seemed to improve growth. SDS-PAGE analysis of cell lysates suggested a notable change in the presence of a few C. crescentus proteins under Coumermycin dosing, one of which is the master regulator SciP. There appeared to be no significant change in accumulations of the other cell cycle master regulators, suggesting supercoiling only regulates a subset of genes. Miller Assays and immunoblots confirmed that a decrease in sciP transcription, leads to less SciP accumulation when gyrase activity is inhibited. We observed similar results were observed in cells treated with another antibiotic and gyrase-inhibitor, novobiocin. We identified plasmid pJC326C, which serves as a reporter for the degree of supercoiling in C. crescentus cells. When DNA supercoiling is inhibited, the plasmid relaxes and migrates more slowly through an agarose gel. We conclude that DNA supercoiling is important in the transcription of at least one cell cycle master regulator in C. crescentus.
Ernesto Castellanos, CSU Los Angeles - Biology
AK4 modulates AMPK and mTOR signalingThe mitochondrial adenylate kinase, AK4, is thought to regulate several biological processes related to the cellular stress response. AK4 expression levels are correlated with cellular survival in response to hypoxia, low nutrient levels, and elevated reactive oxygen species. Reducing AK4 expression by RNA interference also modulates these responses. To identify the mechanisms by which AK4 expression regulates these biological effects, we manipulated AK4 expression levels using small interfering RNAs and assessed core signaling pathways related to cellular stress management. We found that AK4 knockdown enhances both the AMPK and mTOR signaling pathways. AMPK activation serves to enhance cellular redox buffering, autophagy, and re-wire cellular metabolism to promote cell survival under stress. mTOR signaling promotes cell growth and proliferation. The AMPK and mTOR pathways are typically thought to be antagonistic and therefore mutually exclusive. Here, we identify a mechanism (inhibiting AK4 expression) whereby AMPK and mTOR signaling are concurrently activated.
Roland Lacap, CSU Northridge - Biology
Isolation of melanocytes from hair follicles on explant culturesMelanocytes are epidermal pigment cells that produce melanin in the skin. Melanin provides pigment in the skin, usually a tan or brown color. More importantly, it functions to protect skin from environmental risks like prolonged sun exposure. Melanocytes are important to cancer research because it has been a useful model for studying melanoma. In this protocol, our goal is to isolate melanocytes from human hair follicles. With this technique, we extracted human hairs (about 60-70 strands) from donors and explanted them into a primary cell culture. These melanocytes will be used for experimental approaches for studying melanoma. The primary cell culture are still in its early stages, but we expect melanocytes to grow from the outer root sheath (ORS) of the hair follicles.
Ekene Akpati, CSU Northridge - Business and Economics
Nimbus: Actively working towards alleviating global issues like cancer and climate change. (www.nimbus.earth)Nimbus is a platform for discussion, ideation, and collaboration between individuals around the world with vital passions like curing and preventing cancer, ending homelessness, and adopting widespread sustainability. The goal is to develop a hyperactive quasi-think tank of diverse individuals who actively work towards solving pressing global issues. We are researchers, activists, entrepreneurs, and everything in between. We do great things.
Irazema Islas, SDSU - Chemistry/Biochemistry
A Solvatochromic Nucleoside to Report on Point Mutations and DNA ConformationNucleic acids play a key role in not only the storage of the genetic code, but they also regulate the code's expression and play active roles in the metabolic chemistry occurring in cells. Mutations in the genetic code are the key driver of cancer. A variety of methods are available for detecting mutations, including sequencing methods, PCR, and array-based methods, but all of these can be intricate and time consuming. Building on our labâ€™s experience in the design and synthesis of functional nucleoside analogues, we designed and synthesized a tricyclic derivative of cytidine, dinitro-tCO, which we expected to have solvatochromic properties. Studies of solvent effects on dinitro-tCO photophysics show that this brightly colored nucleoside analogue changes its absorbance in response solvation, making it what we believe to be the most solvatochromic nucleoside analogue known. We hypothesize that its solvatochromicity will give it unique capabilities for reporting on point mutations and nucleic acid secondary structures involved in the regulation of gene expressions, such as i-motifs. Our next step is to insert the nucleoside analogue into DNA and measure how its optical absorbance responds to base pairing and stacking. Future work will focus on the application of its solvatochromism to the detection of changes in the conformations and folding patters of DNA and single nucleotide polymorphism.
Kelly Dinh, SDSU - Biology
Characterizing Mutations in Myelodysplastic Syndrome (MDS)Over the years, Myelodysplastic Syndromes (MDS) have been characterized by their genetic mutations within epigenetic regulator genes, but research is still being conducted to gain a better understanding of the cancer. MDS is characterized by clonal hematopoiesis and impaired differentiation which causes progenitor cells to be unable to mature into healthy blood cells leading to a reduction in circulating neutrophils, red blood cells, and platelets. Older adults are at a greater risk and though there are existing treatments, they are not ideal for every patient and non-targeted treatments may result in complications. Even with treatments, one third of patients progress to AML (Acute Myelogenous Leukemia), and in order to acquire better treatments, the mutations are characterized to increase our understanding of the disease. Experiments were conducted with mouse cell lines with mutations in Ezh2, Tet2, Srsf2, and U2af1. By analyzing the characteristics of each cell line, our understanding increased on how these mutations play a role in MDS. As a result, we are now a step forward in understanding MDS.
Yasmeen Nizam and Sa La Kim, CSU Northridge - Biology
Integrin Alpha 1 (ITGA1) Function in Mediating Pancreatic Cancer Cell Resistance to Microenvironment StressThe lack of early detection biomarkers for and therapy-refractory nature of pancreatic ductal adenocarcinoma (PDAC) makes it one of the deadliest. We have previously reported that the cell-surface collagen receptor integrin alpha 1 (ITGA1) is critical for viability, chemotherapy resistance and metastasis of PDAC cells. Proteomic profiling of ITGA1-depleted PDAC cells identified altered levels of endoplasmic reticulum (ER)-resident ATP2A1 and ATP2A3 (SERCA1/3), which are regulators of ER calcium homeostasis and targets of the unfolded protein response (UPR)-inducing drug thapsigargin. Since UPR signaling and ER stress responses activate survival mechanisms in many solid tumor types, we reasoned that ITGA1 upregulation in PDAC may function to suppress SERCA1/3 expression and elevate the basal ER stress response state. In agreement with this hypothesis, SERCA1/3 levels are reduced while GRP78 (ER stress marker) levels are elevated in PDAC tissues and cells. Our preliminary results suggest that ITGA1 is required for resistance to ER stress-induced cell death and upregulation of GRP78. In parallel, we observed that ER stress-induced cell spreading and/or epithelial-to-mesenchymal (EMT) requires ITGA1 expression. Ongoing work aims to investigate ITGA1's role in PDAC cell survival, metastasis and therapy response under more physiologically-relevant microenvironmental stresses.
Kevin Zarghan, SDSU - Chemistry/Biochemistry
The Conserved Structure of the DUF 2419 FamilyThe DUF 2419 superfamily has emerged as a remarkably versatile protein scaffold for the evolution of diverse catalytic activities. The DUF 2419 protein family was recently discovered and thus it is not well understood by the scientific community but some evidence from its orthologs indicates that it is likely involved in tRNA processing. This project is a preliminary effort to structure and function discovery. Here we overexpress the DUF 2419 using both human and bacterial cell lines to produce crystals that can be further analyzed using X-ray crystallography. C9orf64 is a gene located on chromosome 9, that in humans encodes the protein queuosine (Q) salvage protein. Queuosine is a micronutrient modification found on the wobble position of tRNAs. Recent publications indicate that DUF 2419 is involved in the methylation of cell lines in ovarian cancer, breast cancer, colon cancer and acute myeloid leukemia. There is still much to learn regarding its function in the cells of various cancers. In humans the expression of the gene of interest is highest in the duodenum and small intestine but is also expressed in 24 other tissues. The protein was then exposed to JCSG and Salt Rx conditions for a total of 192 separate conditions in both 4 mg/ml and 8 mg/ml concentrations, which are used to generate high quality crystals that can then be further analyzed to confirm the structure and function of DUF 2419 proteins.
Samantha Crawford, SDSU - Chemistry/Biochemistry
Characterization of Resistance Mechanisms to the Dual PI3K and BRD4 Inhibitor SF2523Liver cancer death rates are rising in the United States. Current methods to treat patients with the most common type of liver cancer, hepatocellular carcinoma (HCC) have limited efficacy. Targeting PI3K and BRD4 pathways using the dual inhibitor SF2523 is a novel approach to inhibit the growth of HCC tumor cells. However, chronic exposure of HCC cells to SF2523 results in the development of resistance and sustained cellular growth. We have generated HCC cells resistant to SF2523 using the Huh-7 cell line. These Huh-7 parental and SF2523-resistant cells were used as a model to characterize emerging resistance pathways with a multi-omic approach. Parental and resistant cells were used for RNA-seq to assess transcriptomic changes, ATAC-seq to assess epigenetic changes, phosphoprotein arrays to assess phosphosignaling changes, and small molecule chemical library screens to identify candidate drugs to overcome resistance. This combinatorial approach has identified drug targets to use in addition to the dual PI3K/BRD4 inhibitor SF2523 as a precision medicine approach targeting pathways unique to HCC tumor biology. Such targeted drug combinations are becoming more desirable for HCC as they may be more efficacious and have less toxicity compared to traditional cancer treatments.
Ronelle Caguioa, CSU Northridge - Chemistry/Biochemistry
Can the Atypical G Protein Coupled Receptor ACKR3 (CXCR7) Activate G Proteins During Chemokine Signaling?The atypical chemokine receptor ACKR3 (CXCR7) is a class A GPCR that plays a significant role in tumor growth and cancer metastasis. The activation of ACKR3 has the unique effect of exclusively recruiting β arrestin instead of inducing a G-Protein mediated signaling pathway, however, a handful of studies have proposed that ACKR3 may be capable of coupling and activating G proteins. CXCR4 is another class A GPCR that shares a chemokine ligand, SDF-1 (or CXCL12), with ACKR3, but is known to couple to G proteins. Analysis of the structure and function of these receptors can lead to drug development targeting the migration of cancer cells that utilize ACKR3 receptor for cell movement. We have built the structural models of the complexes of ACKR3 and CXCR4 with either the Gαi protein (GNAI2) or β arrestin (ARRB2). The four resulting complexes are relaxed in their native membrane environment using molecular dynamics (MD) simulations and analyzed in terms of interaction energies between the receptors and their respective signaling partners. The MD simulations reveal that ARRB2 favors coupling to ACKR3 over CXCR4 suggesting that the ACKR3:ARRB2 complex is more stable than the CXCR4:ARRB2 complex. Surprisingly, GNAI2 does not have a strong preference in coupling to either receptor suggesting that biophysically ACKR3:GNAI2 and CXCR4:GNAI2 complexes have comparable stability. This is consistent with experiments that show that ACKR3 can couple to G proteins during chemokine signaling.