2022 Trainee Talks
Zuzana Tirpakova (Terasaki Institute)
Multicellular Hepatocellular Carcinoma Organoids to Define Mechanistic Determinants of Dormancy-driven Therapy Resistance
Hepatocellular carcinoma (HCC) is one of the most aggressive cancers with high recurrence rates. Cancer stem cells (CSCs) play a significant role in cancer recurrence. Anti-proliferative therapies, including Sorafenib, kill the proliferating cells but induce mitotic quiescence in CSCs, which remain residual dormant CSCs. Withdrawal of treatment reactivates the residual CSCs from dormancy, producing new proliferative cancer cells that initiate new tumor formation. Here, we investigate the CSC mechanisms of recurrence, mitotic quiescence, dormancy, and reactivation of CSCs in HCC. Three-dimensional (3D) in vitro multicellular HCC models were developed by co-culture of HCC cells (HepG-2) and stromal stem cells (SSC, C3H/10T1/2). HCC organoids with 190µm diameter and 0.92 circularity index were generated in 3 days of co-culture. A panel of 53 genes related to cancer cell dormancy and reactivation, cancer stemness, cell proliferation, and matrix were evaluated through RT-qPCR. 39 genes were upregulated in a threshold of 2 at -log (false discovery rate), including RASAL1, HIST1H1C, THBS1, CD24 (proliferation and metastasis), NR1D1 (HCC progression), CCNG2 (cell cycle), FN1, ITGA1, ITGA2 (matrix), CD13, HMOX1 (carcinogenesis and drug resistance), EpCAM (cancer stem/progenitor cells), p21, p27, TGFB1 (dormancy). These findings highlight the metastatic microphysiological system fitted with 3D co-culture of HepG2 and SSCs as a critical tool in developing dormant metastatic HCC models.
Audrey Todd (CSU Northridge)
Pancreatic Ductal Adenocarcinoma (PDAC) and Mesenchymal Stem Cell (MSC) 3D Co-Culture May Induce Cancer Cell Dormancy
Pancreatic ductal adenocarcinoma (PDAC) is therapy resistance due to tumor cell dormancy and a dense stromal microenvironment containing mesenchymal stem cells (MSCs). MSCs promote cancer through tumor-stroma interactions. Here a 3D tumor co-culture model is used to study gene expression changes in PDAC cell identity and dormancy. By using human PDAC cells and mouse MSCs, species-specific qPCR primers can evaluate cell specific gene expression profiles. PDAC epithelial-to-mesenchymal transition (EMT) and stemness markers (VIM, CDH1, CDH2, FN1, ZEB1, PEAK1, TDGF1, COL1A2, COL6A3, ITGA1, ITGA2), stress markers (HSPA5), dormancy markers (P27, P16, P21, TGFB1, TGFB2), and recovery markers (SOX9, TGFB3, FOXO3) are analyzed. MSC cancer-related markers (Peak1, Vim, Fn1, Cdh2, Col1a2, Col6a3, Inhba, Vcl, Snai2, Acta2, Tdgf1, Rai14) are also examined. When in 3D co-culture, PANC1 cells showed significantly higher expression of CDH1, VIM, CDH2, FN1, HSPA5, TGFB2, and SOX9 relative to monoculture. This suggests a mix of epithelial and mesenchymal phenotypes, higher levels of stress, and that some cells are dormant while others recover from dormancy. In 3D co-culture the C3H cells showed significantly higher expression of Peak1, Col6a3, Inhba, Vcl, Acta2, and Tdgf1 relative to monoculture, suggesting a stemness phenotype, increased ECM secretion, and promotion of EMT within the PANC1 cells. Further study of this model will aid in understanding how tumor cells evade cancer therapies.
Eduardo Hernandez (CSU San Francisco)
Investigating the Interaction of Kaposi’s Sarcoma-Associated Herpesvirus Viral G Protein-Coupled Receptor and Angiogenic Proteins
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the causative agent for Kaposi’s sarcoma (KS). KS is the most frequent cancer in males and children in the Mediterranean and African countries. The virus encodes a constitutively active signaling molecule called the viral G protein-coupled receptor (vGPCR) which is critical for the initiation and progression of KS. KSHV establishes lifelong infection of the human host and currently there is no treatment. A previous study’s proteomics screen revealed that vGPCR is suggested to interact with many human proteins. Among the interactions associated with KSHV are two understudied angiogenic proteins called endoplasmic reticulum membrane complex subunit 10 (EMC10) and mannoside acetylglucosaminyltransferase 5 (MGAT5). However, these vGPCR interactions with host proteins and the downstream implications have yet to be explored. My study investigates the potential interaction between vGPCR and these specific angiogenic proteins. I hypothesize that KSHV vGPCR interacts with EMC10 and MGAT5 and that these interactions modulate angiogenesis. Immunofluorescence assays and western blotting were used to analyze angiogenic protein expression in KSHV vGPCR-expressing cells. Co-immunoprecipitation will also be used to validate the interactions. This study will provide information on understanding viral-host protein interactions affecting the modulation of angiogenesis and can lead to therapeutic targets for KSHV infection.
Jennifer Waters (CSU San Diego)
Omental Preadipocytes Support Ovarian Cancer Tumorigenesis by Mediating Extracellular Matrix Reorganization by Cancer Cells
Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty tissue that lines the abdominal wall. We hypothesize that preadipocytes, the precursor cells that give rise to omental adipocytes, are important mediators of cancer progression in the omental tumor microenvironment. To evaluate this hypothesis, we tested whether preadipocyte secreted factors altered tumorgenicity of ovarian cancer cells using colony formation and cell viability in vitro and a limiting dilution in vivo in a subcutaneous mouse model. The mouse studies showed that low dilutions of cancer cells require preadipocytes for tumor formation and that transplanted cancer cells require preadipocytes to sustain this phenotype. In vitro assays revealed that preadipocyte secreted factors increase cancer cell clonogenicity and viability. To identify signaling pathways induced by preadipocytes we co-cultured cancer cells with either preadipocytes or mature adipocytes and performed RNA sequencing. We identified differentially expressed genes in cancer cells that were unique to preadipocyte co-culture, including DCN, MMP2, COL6A2 and COL12A1, and IGFBP5; all genes involved in extracellular matrix organization. Our findings highlight the role of preadipocytes in modulating the ovarian tumor microenvironment, and understanding these processes will enable the design of more effective therapies for treating ovarian cancer.
Farhana Runa (CSU Northridge)
RAI14 Promotes PDAC Progression, Associates with the PLK1/NEK6 Pathway and Correlates with Poor Outcomes
Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal solid tumor estimated to be the second-leading cause of cancer-related death in the United States by 2030. The intrinsic resistance PDAC to chemotherapy is governed by cell-/patient-level heterogeneities and desmoplasia. We previously reported that PEAK1(Pseudopodium-enriched atypical kinase1) is a pseudopodium-enriched (PDE) protein co-expressed with integrin α1 (ITGA1) that promotes cancer metastasis. Here, we report on the role for another PDE and PEAK1 co-expressing protein – Retinoic Acid Induced 14 or RAI14) in PDAC progression. Importantly, RAI14 knockout in PDAC cells abrogated their ability form tumors and metastasize in the Gallus gallus CAM (Chorioallontic Membrane) assay. Moreover, using a RAI14-focused bioinformatics workflow, we discovered that RAI14 may cooperate with the PLK1/NEK6 pathway to control mitotic progression. In support of this possibility, survival analyses of PDAC patients showed that significantly worse outcomes in were associated with high RAI14 expression. These findings improve our understanding of the molecular control for PDAC and provide a new mechanistic understanding of PDAC progression that may be leveraged to improve patient outcomes.
Mikella Robinson (CSU San Diego)
TWEAK-Fn14-RelB Signaling Cascade Promotes Stem Cell-Like Features that Contribute to Post-Chemotherapy Ovarian Cancer Relapse
High Grade Serous Ovarian Cancer (HGSOC) is the most lethal gynecological disease, with 70% of patients relapsing within 18 months. Disease recurrence may be due to cancer stem-like cells (CSCs) that are resistant to chemotherapy and capable of reestablishing heterogeneous tumors. We previously showed that CSC maintenance involves activation of alternative NF-κB transcription factor, RelB, however the mechanism is unknown. We hypothesize that TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine important for tissue repair, and its receptor, Fn14, contributes to CSC development following chemotherapy by activating RelB. We found that TWEAK and Fn14 are enriched in ovarian tumors following chemotherapy treatment and are strong inducers of RelB. TWEAK significantly enhances spheroid formation, asymmetric division capacity, and expression of stem cell maintenance gene, SOX2, and epithelial-mesenchymal transition genes VIM and ZEB1 in HGSOC cells. These phenotypes were significantly enhanced when TWEAK is combined with carboplatin and were significantly inhibited with CRISPR knockout of RelB. Blocking the TWEAK-Fn14-RelB signaling cascade with a small molecule inhibitor of Fn14 prolonged survival following carboplatin treatment in a mouse model of HGSOC. These data provide new insights into ovarian cancer CSC biology and highlight a signaling axis that can be harnessed for therapeutic development.
Brandon Wong (CSU Los Angeles)
Prediction of Four Acidic Amino Acid Residues within t-Darpp that are Critical for Binding to Calcium
Intracellular calcium ions maintain a variety of functions as chemical regulators and messengers. Some proteins that bind to free calcium ions have been linked to tumor progression and survival after treatment in several types of cancers. Overexpression of the truncated isoform of Dopamine and cAMP-regulated phosphoprotein 32, t-Darpp, causes resistance to trastuzumab (Herceptin), a frequently used biologic used to treat HER2+ breast cancer. Through chemical modification and in vitro binding calcium binding studies we show that carboxyl groups within t-Darpp are required for binding to calcium and that binding is specific for calcium because it is unable to be competed-offfrom t-Darpp by treatment with other divalent metals Zn2+ and Ni2+. Multiple sequence alignment of t-Darpp with 88 vertebrate orthologs identified 11 Asp and Glu positions within t-Darpp that are evolutionarily conserved. We utilized the metal ion binding prediction software program IonCom to identify which of these conserved residues are also potential calcium binding sites. IonCom predicted seven Asp and Glu residues that may be critical for binding to calcium, four of which are also conserved. Combined with our ortholog analysis, we predict that Asp70, Glu74, Glu77, and Asp100 in t-Darpp are critical for binding to calcium. We have mutated these four residues by substitution with Ala and are currently performing experiments to determine if they are required for calcium binding.
Joseph Alas (CSU Pomona)
Studying the Effects of Genistein on the Yeast Cell Cycle
One of the leading causes of morbidity and mortality in the world is cancer. The most frequently diagnosed cancer types worldwide are lung, breast, and colorectal cancer. While many chemotherapeutics and anticancer drugs have been developed, they do not work on all types of cancers and can have unwanted side effects. Therefore, studying alternative agents is important. Genistein, a phytoestrogen found in soybeans, is known to lower the risk of cancer, though limited research has been done to show this. Genistein is shown in in vitro studies that many human cancer cell lines with small amounts of genistein can induce the apoptosis mechanism. Few in vivo studies show genistein is effective as an alternative cancer treatment. Genistein causes G2/M arrest and can override the G1/S phase arrest in human cancer cells. My research project uses Saccharomyces cerevisiae to study the cell cycle. We will use hydroxyurea to cause a cell cycle arrest and then use genistein to see how it can override the cell cycle. We will determine phases of the yeast cells with microscopy and plan to do FACS analysis to show the phases of the yeast cells by DNA content. We will then use western blot to identify which proteins are being upregulated/downregulated when the override mechanism has occurred. We are looking to see, if in yeast, the G2/M arrest is induced and/or the G1/S phase override occurs. So far, we only see the override of the G1/S phase arrest.
Gabriela Ortiz-Soto (UCC)
Involvement of the Oncoprotein Metadherin in Inflammatory Breast Cancer Progression
Inflammatory breast cancer (IBC) is one of the most lethal subtypes of breast cancer (BC) that accounts for approximately 2-4% of all cases of BC. The lethality of IBC originates from its nature of invading the vascular and lymphatic systems and from the absence of a typical tumor mass, resulting in an accurate and early diagnosis, as well as the development of effective targeted therapies. Our previous studies identified Metadherin (MTDH) overexpression, a cell adhesion molecule, in IBC cells and patient tissues. MTDH has been found to play a role in signaling pathways related to cell survival, proliferation, migration, and invasion in other types of cancer. However, its mechanism of action and role in the progression of IBC remain unknown. Therefore, the objective of this study is to assess the functional role of MTDH in IBC. To evaluate MTDH function, IBC cell lines, SUM-149 and SUM-190, were edited with CRISPR/Cas9 vectors for in vitro studies. We evaluated MTDH expression by qPCR and immunoblotting that confirmed reduced MTDH levels in edited IBC cells. Our preliminary results demonstrated that after editing, IBC cell proliferation, viability, migration, invasion, and spheroid formation were significantly reduced. Furthermore, immunoblot results showed a significant reduction in total STAT3 in SUM-149 cells, suggesting a possible role for MTDH in the regulation of STAT3 signaling. These results further suggest that MTDH plays a potential role in the progression of IBC.
Elizabeth Ortiz (CSU Northridge)
Morphology and Expansion of HER2-Positive Breast Cancer Patient-Derived Organoids
Patient-derived organoids (PDxO) are 3D cell culture models that have preserved tumor-stroma interactions, tumor heterogeneity, tumor drug resistance, and metastatic tumor properties. These qualities allow PDxOs to yield responses to cancer therapies that parallel in vivo tumor responses. HCI-005 and HCI-012 are both HER2+ PDxO lines of metastatic ductal carcinoma origin. In contrast, HCI-005 is estrogen receptor and progesterone receptor positive. After finding that matrigel provides a better 3D environment for organoid development than biogel, it is necessary to expand and note morphological differences between these HER2+ PDxOs. Differential interference contrast microscopy shows that HCI-005 forms an asymmetrical quadrilateral shape but becomes spherical after 60 hours(hrs), and it reaches a radius of 100µm at 200hrs. HCI-0012 organoids group into small clusters that fuse to form oval organoids with a defined edge after 60hrs. This morphology yields radii ranging from 100µm for standard oval-shaped organoids to 160µm for long organoids by 200hrs. The most striking morphological difference between these PDxOs is the formation of invasive protrusions. HCI-005 forms small protrusions measuring 20µm by 110hrs. After 70hrs, HCI-012 form protrusions that reach a length of 200µm that later fuse to neighboring organoids at 110hrs. These morphological observations are essential for determining the quality of invasiveness and changes to organoid growth by cancer therapies.
Omar Lujano (CSU San Diego)
Chemotherapy Induced Activation of TWEAK-Fn14-NF-kB Signaling Axis Enhances Adhesion via Integrin Expression
Ovarian cancer is the most lethal gynecological disease in the United States, resulting in over 15,000 deaths annually. Despite a strong initial response to platinum/taxane–based chemotherapy, relapse occurs in about 80% of patients within 24 months. Tumor recurrence is likely due to cancer stem-like cells (CSCs), a small population of cells that are resistant to chemotherapy and colonize metastatic sites. There are several gaps in our understanding of how these cells facilitate disease relapse and what role the tumor microenvironment (TME) plays in this process. Thus, there is an urgent need to clarify ovarian CSC mechanisms for the identification of therapeutic targets. Based on our previous work, I hypothesize that chemotherapy induced activation of the TWEAK-FN14-NF-kB signaling axis drives the development of CSCs with improved adhesion. We demonstrate that TWEAK, a cytokine and its receptor, Fn14, are enriched in the TME following chemotherapy. Stimulation of ovarian cancer cells with TWEAK induces activation of NF-kB and the induction of stem-like properties. Taken together these findings show a potential role for chemotherapy in the development of CSCs. Furthermore, the TWEAK-Fn14-NF-kB axis also induces expression of clinically relevant integrins including ITGA5 and ITGAV. Importantly, the significance of the study aims to uncover a unique integrin-adhesion mechanism for ovarian cancer relapse driven by TWEAK-Fn14-NF-kB signaling in CSCs following chemotherapy.
Nikolas Yousefi (CSU Northridge)
The Role of CXCR4 and CXCR7 in Melanoma Metastasis and Migration
Stromal cell-derived factor 1 (SDF1), also known as CXCL12, is a type of chemokine, a signaling protein able to induce chemotaxis, that has been extensively studied for its role and presence in cancer and more specifically, melanoma. The two SDF1 receptors are CXCR4 and CXCR7. Our aim was to look at the role of CXCR in melanoma migration by silencing CXCR4 and CXCR7 in B16-F10 murine melanoma. A classic chemokinesis assay is the Boyden chamber migration and chorioallantoic membrane (CAM) assay looks at metastasis in chicken embryos. I used these two to assess the role of CXCR4 and CXCR7 in melanoma after siRNA knockdown. Results from the Boyden chamber experiments showed that knocking down CXCR4/7 significantly decreased migration. On average, the number of migrated cells in CXCR4 and CXCR7 transfected cells was shown to have decreased by about 48% and 60% respectively. I also observed that melanoma cells decreased their cell area after CXCR4 or CXCR7 siRNA KD and CXCR4 KD melanoma showed more stress fibers. In conclusion, I determined that knocking down CXCR4 or CXCR7 played a role in melanoma migration, by reducing their motility as well as altering melanoma cell morphology. These findings are a step towards finding a possible pathway to treat patients afflicted with melanoma before it can metastasize. Future experiments using CAM assays will determine if knocking down CXCR4 and/or CXCR7 will hinder melanoma metastasis.
Kassy Lopez (City of Hope)
Methylglyoxal-induced Genomic DNA Adducts are Mutagenic and are Present in Triple Negative Breast Cancer
Methylglyoxal (MG) is a reactive by-product preferentially formed from glycolysis and covalently modifies DNA, RNA, and protein forming advanced glycation end products (AGEs). We have observed elevated MG-AGEs in patients with diabetes and cancer, implicating an association between AGEs and disease pathology. DNA is an important target for MG modification, as these adducts may lead to mutations that can drive cancer onset and progression, but its exact mutational signature in the genome remains unexplored. A factor that can influence MG-induced mutagenesis is cellular repair of the damage. We hypothesize that MG induces genomic mutations and decreased DNA repair capacity will increase mutagenesis. To explore this, we utilized cells deficient in nucleotide excision repair (NER) and base excision repair (BER) (CRISPR-Cas9 KO of XPA and APE1, respectively). Using a hypoxanthine phosphoribosyl transferase (HRPT) forward mutation assay, we found that MG increases mutation frequency and DNA adduct formation, which is further exacerbated in cells deficient in NER and BER. We also found that MG-DNA adducts are increased in triple negative breast cancer compared to surrounding tissues, suggesting an association with aggressive cancer development. Given that decreased DNA repair capacity increases susceptibility to cancer, MG-AGES may serve as a biomarker for cancer progression in diabetic patients with elevated MG due to hyperglycemia.
Alejandro Gomez (CSU Northridge)
Gelatin Methacryloyl and Laponite bioink for 3D bioprinted Multicellular Organotypic Tumor Modeling
Three-dimensional (3D) in vitro tumor models capturing the pathophysiology of human tumors is essential for cancer biology and drug development. However, it is still challenging to simulate the tumor microenvironment (TME) due to its heterogeneity. Here, we developed a nanoengineered ion-covalent crosslinkable bioink containing pancreatic cancer cells (MIA PaCa-2) and stromal cells (C3H10T1/2) to construct 3D multicellular organotypic tumor models. The bioink composed of gelatin methacryloyl (GelMA) was designed to mimic the extracellular matrix (ECM) of TME. High GelMA (10%) with low Laponite (1.0-2.0%) composite hydrogels showed good mechanical properties for 3D printing and supported cell viability and proliferation. Increased Laponite ratios induced cell aggregation to form larger 3D tumor structures. In qPCR experiments, the Laponite ratio increased the upregulation of growth factor and tissue remodeling-related genes in tumor cells, whereas cell cycle and proliferation-related genes were downregulated. On the other hand, in the case of cancer-associated fibroblasts (CAFs), an increase in the Laponite ratio indicated an overall upregulation of the mesenchymal phenotype-related genes. Our study provides a rationale for using GelMA/Laponite-based hydrogels as 3D cancer modeling bioinks. Furthermore, we expect that our 3D bioprinted multicellular organotypic tumor models will pave the way for developing high-throughput drug screening platforms.
Kinsey Nelson (CSU San Diego)
VAX014 Activates Innate Immune Ssensing Mechanism(s) to Overcome Immune Escape in Cancer
As the search for more effective strategies to treat cancer advances, recent insights highlight the availability of tumor antigens, antigen presentation, T cell activation and Type I interferon (IFN) as critical steps to develop immune-mediated control of tumors. Tumors adapt to escape immune control by, for instance, overexpressing immune checkpoint molecules (e.g., PD-L1), downregulating MHC-I, and dysregulating Type I IFN production. Oncolytic therapies can promote antigen release and cross-presentation, activate anti-tumor immunity and work with current checkpoint inhibitors. VAX014 is a novel bacterial minicell-based oncolytic immunotherapy recently demonstrated to enhance survival in the MB49 mouse model of bladder cancer. In that work, MB49 tumors upregulated PD-L1 when treated with VAX014, and the combination of VAX014 with systemic PD-L1 blockade promoted better local and systemic anti-tumor immunity and survival. In this study, we demonstrate that VAX014 triggers production of Type I IFN in MB49 cells (p<0.0001) leading to upregulation of both PD-L1 (p<0.0001) and MHC-I (p<0.0001) expression. Additional mechanistic studies suggest that VAX014 activates multiple innate signaling pathways, including STING and/or RIG-I, to induce production of Type I IFN. Taken together, these results suggest that VAX014 therapy may help overcome several mechanisms of immune escape in cancer, leading to better outcomes for patients.
Blake DuPriest (CSU San Jose)
Increased Intracellular pH Promotes Cell Death in the Developing Drosophila Eye
Studies have investigated the proteins and signaling pathways regulating cell death, however much less is understood about the chemical signals that regulate cell death. Cells generate acids as a result of normal cellular processes, and acid levels inside cells, or intracellular pH (pHi), can change in response to certain stresses or cues. Compared to normal cells, cancer cells have an increased pHi. This increase in pHi is thought to contribute to disease progression by altering cell behaviors: increasing proliferation, reprogramming metabolism and decreasing cell death. Recently developed Drosophila models, wherein over-expression of the ubiquitous sodium-proton exchanger DNhe2 (homolog of mammalian NHE1), showed increased pHi, increased cell proliferation, and aberrant patterning. We predicted increased pHi in our system would inhibit cell death, however examination of adult fly eyes showed decreased overall size. Cell counts of the fly eye revealed a significant decrease in the number of interommatidial lattice cells at the end of pattern formation with over-expression of DNhe2. This is despite increased proliferation reported with over-expression of DNhe2 larval eye discs. Preliminary data shows that cells may be eliminated by autophagy at increased pHi, and that this may be regulated by the oncogene Myc. Together, our findings elucidate mechanisms for pH-regulation of conserved, critical developmental processes and provide evidence for new paradigms in growth control.