EVALUATING THE ROLE OF NUTRIENTS AND NUTRIENT METABOLISM IN CANCER PREVENTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013703
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Nutrition

Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: human nutrition.Although our genes influence our risk of cancer, most of the difference in cancer risk between people is due to factors that are not inherited. People who are overweight or obese are associated with an increased risk of cancer, including pancreatic cancer. Unfortunately, there is still limited information on how certain nutrients/diets or nutrient metabolism provide their health benefits, their mechanism of action and biological targets. The long term goal of my laboratory is to elucidate the effect of diet during cancer progression. In particular, this project will focus on evaluating the role of lipid metabolism in pancreatic cancer progression. Pancreatic cancer is a deadly disease with unsatisfactory treatment options. The poor prognosis of pancreatic cancer requests the exploration of novel drug targets. Phospholipase D1 (PLD1), a lipid-signaling enzyme, has been shown to play a role in the progression of various types of cancer; however there is no information about the role of PLD1 in pancreatic cancer. Preliminary studies in our laboratory have shown that inhibiting PLD1 reduces pancreatic tumor growth, extends survival and enhances the chemotherapeutic effect of gemcitabine in animal models of pancreatic cancer. These data suggest that PLD1 may play a role in pancreatic carcinogenesis. The aim of this project is to evaluate the role of PLD1 in pancreatic cancer to find out whether PLD1 can be a useful new target for pancreatic cancer treatment. We will employ biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance PLD1 as a novel target. At the completion of these studies, we expect to have deciphered key functions of PLD1 in pancreatic cancer. Results from the proposed work will provide new information on the effects of a lipid-signaling enzyme on pancreatic tumorigenesis. Given the importance of pancreatic cancer and the lack of effective agents against it, we believe that the proposed work will advance the understanding of how lipid metabolism influences this deadly disease that affects thousands of families in California and in the United States.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	3840	1030	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1030 - Cellular biology;

Keywords

pancreatic cancer

lipid-signaling

phospholipase d

pancreatic carcinogenesis

cancer prevention

obesity

diet

Goals / Objectives
Environmental factors, including certain types of diets, are associated with an increased risk of cancer.Obesity is a major health concern in California and in the US. Many factors may explain why people become overweight or obese, including genetic, hormonal, environmental, emotional, and cultural factors. It is estimated that 50% of major current health concerns, including obesity, type-2 diabetes and cancer, can be prevented by changes in dietary habits.Although our genes influence our risk of cancer, most of the difference in cancer risk between people is due to factors that are not inherited. People who are overweight or obese are associated with an increased risk of cancer. In particular, people who are overweight or obese are about 1.5 times as likely to develop pancreatic cancer as normal-weight people. However, to date, there is still limited information on which are the specific components in the diet that provide their health benefits, their mechanism of action, biological targets, metabolism, individual genetic and biological modifiers of absorption/effects. The long term goal of my lab is to elucidate the effect that diet have during cancer progression. Therefore, in this project, we will focus on evaluating the role of lipid metabolism in pancreatic cancer progression.Pancreatic cancer is a dismal disease in need of novel therapeutic targets.Pancreatic ductal adenocarcinoma (PDA) is a complex and lethal cancer with a five-year survival of < 7.9%. Surgery, which offers the only realistic hope, has a limited role, whereas current chemotherapy or radiation therapy regimens offer minimal or no help. Thus, there is an urgent need for new strategies to combat this disease; being the identification of new therapeutic targets a critical component.Phospholipase D1 signaling regulates tumor growthThe lipid signaling enzyme Phospholipase D (PLD) catalyzes the conversion of the membrane phospholipid phosphatidylcholine to choline and phosphatidic acid (PA). PLD has a double role in the cell: phospholipid turnover: maintenance of the structural integrity of cellular/intracellular membranes, and cell signaling through PA and its metabolites. Indeed, through PA, PLD has been implicated in a variety of physiological cellular functions, such as intracellular protein trafficking, chemotaxis of leukocytes and cell proliferation.Elevation of either PLD1 or PLD2 (two mammalian isoforms of PLD) contributes to cancer progression. For example, PLD1, increased in abundance or activity in various human cancers, is linked with proliferative signaling and resistance to cell death. In particular, PLD1 is elevated in clinical samples of PDA, and positively correlates with vascular invasion and poor prognosis. Interestingly, PLD1, but not PLD2, in the tumor environment promotes tumor growth and metastasis. Furthermore, ablation of PLD1 in the tumor environment compromises the growth of lung and skin tumors. These findings suggest the potential use of PLD inhibitors as cancer therapeutics. However, to date, there is a dearth of information regarding whether PLD1 plays a mechanistic role in pancreatic cancer.Thus, given the correlation between PLD1 expression and aggressive features of PDA, we explored whether PLD1 may be implicated in PDA growth. In preliminary studies, we found that PLD1 ablation reduced pancreatic tumor growth, extended survival, and enhanced the chemotherapeutic effect of gemcitabine in murine models of pancreatic cancer.OBJECTIVES: Based on the above, the long-term goals of this project are to understand the role of lipid metabolism in PDA pathogenesis, chemoprevention, and therapeutics. Specifically, the objective of this proposal is to define the role of the lipid-signaling enzyme PLD1 in PDA growth. We hypothesize that PLD1 is a key enzyme in PDA growth. We will employ biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance PLD1 as a novel therapeutic target. Accordingly, the objectives of this specific project are to:1. Determine the role of PLD1 action in regulating PDA progression2. Characterize the mechanism of action of PLD1 action in regulating PDA progression, and3. Establish whether PLD1's role in PDA growth is critical in tumor cells and/or the microenvironment.At the completion of these studies, we will have defined the role of PLD1 in PDA. If our hypothesis is correct, the results will establish PLD1 as a novel and heretofore unappreciated major regulator of PDA progression with potential for therapeutic manipulation.

Project Methods
Methods Objective #1: We will determine the role of PLD1 in regulating PDA progression by: a) defining the in vivo role of PLD1 in the development of pancreatic cancer using genetically engineered mouse models; and b) identifying the molecular mechanisms underlying the inhibition of PDA progression by disruption of PLD1. We will address these goals as follows:a) Define the in vivo role of PLD1 in the development of PDA using genetically engineered mouse modelsWe will use a mouse model of early stage PDA by conditionally expressing an endogenous KrasLSL-G12D allele in developing pancreatic tissues through the use of pancreas-specific Cre recombinase alleles. KrasLSL-G12D; Ptf1aCre/+ ("KC") mice reproducibly show metaplasia and PanIN formation beginning at ∼7weeks of age, with progression to PDA at ∼1 year, contain ductal lesions that mirror human PanINs, and stochastically develop metastatic PDA. We have crossed these mice to the Pld1-/- mice and generated the KC; Pld1-/- mice.To evaluate the role of PLD1 in tumor growth rate, we will conduct bimonthly imaging of KC and KC; Pld1-/- mice (12 mice per group, per time point) using PET/CT with 18FDG, as previously shown. In addition, mice will be euthanized at 5 months (early) and at 10 months of age (advanced), to evaluate the degree of progression (e.g., from pre-invasive to invasive PDA), and the degree of recruitment of the tumor microenvironment. Cellular proliferation and the activation of characteristic signaling pathways in PDA [MAPK, EGFR, mTOR, known to be associated with PLD1, will also be assessed by Immunohistochemistry and Western blot.Methods Obejective # 2Identify mechanisms that suppress PDA progression when PLD1 is disruptedWe will perform next generation sequencing analysis (i.e., RNA-seq) to obtain insight into the mechanisms underlying the impairment of PDA progression by PLD1 ablation. These data will be generated from the animal tissues of the previous sub-aim. Knowledge of genetic and gene-expression changes associated with PDA initiation and progression can facilitate advancements in early detection as well as provide novel targets for therapeutic intervention. Importantly, the substantial toxicity noted in a recent clinical trial simultaneously targeting the MAPK and PI3K Ras effector pathways emphasizes the need to identify novel therapeutic avenues for PDA.Molecular characterization of KC; Pld1-/- pancreatic cancer samplesRNA isolated from KC and KC; Pld1-/- PDA tumor tissues will be assessed for quality using an Agilent Bioanalyzer. Only RNA samples with RIN values greater than 8.0 will be used for sequencing. Poly-adenylated RNA will be enriched from total RNA samples and subsequently used to generate stranded mRNA-seq libraries using a KAPA Stranded mRNA-Seq kit (Kapa Biosystems). Libraries will be assessed by Bioanalyzer and sequenced at the Cancer Center's Genomics Shared Resource using an Illumina HiSeq 4000 sequencing system. Up to 8 RNA-seq libraries will be pooled per flow-cell lane to ensure adequate sequence coverage for expression analysis (>30 million reads). Sequencing data will be analyzed with help from the Bioinformatics Shared Resource. Data will be aligned to the human genome reference assembly and transcript abundance will be called using the RSEM program. Differentially expressed genes will be identified using the DESeq program. Pathway analysis programs such as Gene Set Enrichment Analysis (GSEA) and The Database for Annotation, Visualization and Integrated Discovery (DAVID) will be used to identify biological pathways deregulated in in these samples.A power analysis was done using a calculation designed for RNA sequencing experiments to determine the feasibility of our RNA sequencing experiments. Assuming that we will average 40 reads per gene and will have a coefficient of variation around 0.5 (estimated from a previous RNA sequencing experiment done by our collaborator Dr. Jennie Williams at Stony Brook University), using an alpha of 0.05 and a power of 90%, using 6 samples per group (KC vs. KC; Pld1-/-) will allow us to reliably measure genes with a fold-change of 2.1 or greater.Expression changes identified in our dataset will be confirmed using qRT-PCR analysis, and changes will also be validated at the protein level by immunohistochemistry. The pathways found to be enriched in KC; Pld1-/- mouse samples will be compared to expression analyses of KC samples to distinguish differential corruption of biological processes. To confirm these findings, we will perform functional studies in human PDA cells in culture to assess the role of any identified key genes and/or pathways. Expected results, potential problems and alternatives: We anticipate the animal studies to document a key role of PLD1 in PDA growth. We have already established the KC; Pld1-/- mice and we have experience with transgenic models of PDA. Therefore, we expect no significant technical difficulties conducting this work. Besides the KC model, other important and complementary animal models of PDA are LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, in which additional mutations in tumor suppressor genes are incorporated to generate robust mouse PDA models for the exploration of medical applications and tumor biology. Due to time restrictions, we propose to defer for a future proposal the use of KPC and KPC; Pld1-/- mice.We expect to identify many altered genes and pathways with RNA-seq profiling. As such, we may need to focus our initial validation efforts on those genes and pathways that are most plausible (most well established) in order to more quickly advance understanding of the PDL1 mechanism of action. Alternatively, focusing on novel genes and/or pathways may open up entirely new areas for study and development of therapeutics that will potentially help PDA patients throughout the world.Methods Objective #3: We will define whether PLD1's role in PDA growth is critical in both tumor cells and in the microenvironment by defining the in vivo and cellular roles of PLD1 in regulating cell growth in pancreatic cancer. We will explore whether PLD1 either in tumor cells or in the microenvironment is critical for PDA growth by performing two tumor progression studies of orthotopic PDA xenografts as follows:· To determine if PLD1 in the tumor affects cellular behavior, human Panc-1 pancreatic cancer cells expressing endogenous PLD1 levels (this cell line highly expresses PLD1, or Panc-1 cells in which PLD1 has been knockout (Pld1-/- cells), will be orthotopically transplanted into immunodeficient mice.· To dissect whether PLD1 in the microenvironment is critical in pancreatic cancer growth, we will orthotopically implant mouse KPC cells (luciferase expressing) in immunocompetent WT and Pld1 -/- mice.For both approaches, tumor progression will be monitored using the IVIS system for chemiluminescence activity. Cohorts of mice (12 mice/group) will be euthanized at 1 and 3 months to examine early and advanced disease stages. Cellular proliferation and the activation of characteristic signaling pathways will also be quantified. In particular, we will evaluate if PLD1: a) stimulates Ras activation to increase proliferation; and b) generates survival signals that suppress apoptosis. These approaches will define whether PLD1 in tumor cells and/or in the microenvironment affects PDA tumor growth.End points: tumor weight; gross metastatic incidence (liver, lymph nodes, ascites, liver weight); Animals will be euthanized and the pancreas/tumor excised, weighed and portions of them saved in formalin and liquid nitrogen.Anticipated results and interpretation: We anticipate the animal studies to document a key role of PLD1 in PDA growth and enable us to determine whether PLD1 plays a critical role in the tumor and/or the tumor microenvironment. Given our experience with PDA orthotopic models, we expect no difficulties conducting this work.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The main outreach of my research has been through the publication of original research articles, as well as seminars and talks on the topic and nutrition and cancer are given at scientific congresses that can reach physicians, other health care providers, public health professionals, and to the general population through general public publications, as well as California cancer patients and their families. Furthermore, the target audience of my educational efforts have been minority students directly benefit from the participation of the summer educational and research experiences. Finally, target audience also included prospective students and their families, as well as community and industry stakeholders with whom I interacted at the CA&ES booth during the 2020 World Ag Expo. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this review period, I have mentored or currently mentoring 2 Ph.D., 3 M.S. students, a visiting graduate student and 12 undergraduate students. One M.S. student has graduated, and I am currently advising as chair 2 Ph.D. students and 2 M.S. students. With the visiting graduate student, we have published 5 research papers during this review period and she has now accepted a faculty position. In particular, I am actively involved in mentoring underrepresented students in scholarship, research, and creative activities. In the last two years, I provided mentoring support to students from the CURE program, the NSF LSAMP/CAMP program and currently the PREP program. For example, the UC Davis CURE Program addresses the pressing need for a diverse cancer research community that reflects the nation's heterogeneity, and is sensitive to the significant disparities in cancer health across diverse populations. As a mentor in the CURE program, I have mentored Ms. Yasmin Esparza, who completed her Biochemistry degree in June 2020. This last year, I also mentored Ms. Jazmin Machuca, who participated in the NSF LSAMP/CAMP program. It is important to note, that both Yasmin and Jazmin are co-authors of a research paper recently accepted for publication (EGCG sensitizes chemotherapeutic-induced cytotoxicity by targeting the ERK pathway in multiple cancer cell lines; Archives Biochem. Biophysics, 2020: See publication # above). I have always considered that the experience of an undergraduate student in a research laboratory can be of immense value in their future career decisions, and in their understanding of the scientific thinking. In addition, I have been a member of three dissertation committees, three qualifying committees, and one preliminary exam committee. I have also participated in mentoring international students. For example, I mentored two MS students from the AgroSup Dijon University (France), who worked remotely (due to COVID-19) in 2020, completed a research project and wrote a thesis with the obtained result, in order to obtain their professional degrees of Engineers in Food Science. Their supervision in all the steps (experimental and analysis/writing, through zoom meetings) implied a significant commitment of my time. I also trained and worked with Ran Wei, a visiting graduate student from Zhejiang University, who spent 2 years in the lab completing her research work in my laboratory for graduation. Of note, we have published several manuscripts during her visit. Of note, Ran returned to China, defended her doctoral thesis and she now secured a faculty position in the area of tea science research. These visits have a direct impact on the career advancement of the international visitors. It is noteworthy that they also provide graduate and undergraduate students in my laboratory the possibility of interaction with highly motivated and trained scientists/students, and open their minds to international nutritional concerns that are also of relevance locally. How have the results been disseminated to communities of interest?We have disseminated our research through: a) The presentation of several abstracts/posters to national and international conferences. b) Publication of research papers. c) Teaching undergraduate courses on nutrition and health at UC Davis. d) Supporting the career advancement of minority students. I have been and I am a mentor for a student participating of the UC Davis Continuing Umbrella of Research Experiences (CURE) program (Ms. Yasmin Esparza), which provides research training for diverse UC Davis undergraduates in their junior and senior years. I am the Director of the NUTGAP program and I am mentor of NSF LSAMP/CAMP program (Ms. Jazmin Machuca), and the PREP program (Ms. Brandy Weathers). e) Participation in the 2020 World Ag expo CA&ES booth on February 12. I had an opportunity to inform participants about the exciting research being performed in my lab and in the College, hand in brochure and information about the college, UC Davis and Davis to numerous prospective students and their families, as well as community and industry stakeholders. What do you plan to do during the next reporting period to accomplish the goals?I will continue the ongoing projects focused on: A) the understanding the role and mechanism of action of the lipid-signaling enzyme phospholipase D1 (PLD1) in pancreatic cancer growth; B) investigating the role of high fat diets (obesogenic diets) in pancreatic cancer; and C) investigating the anticancer effects of phytochemicals present in US agricultural products in certain cancers. In addition, I will continue to actively be involved in mentoring of our next generation of scientists.

Impacts
What was accomplished under these goals? The long-term goal of my research group is to elucidate the effect that diet and select dietary components have on cancer progression. During the current period we have worked on the following two projects: A- One major aspect of my research interest is to define the role of the lipid-signaling enzyme phospholipase D1 (PLD1) in pancreatic cancer growth. We hypothesize that PLD1 is a key enzyme in PDA growth. This is a work in progress. We will employ biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance PLD1 as a novel therapeutic target. Pancreatic cancer (PC) is a deadly cancer with unsatisfactory treatment options. The poor prognosis of PC requests the exploration of new ways to treat this disease. Phospholipase D1 (PLD1) is a lipid-signaling enzyme that has been shown to play a role in the progression of various types of cancer. However, there is no information about the role of PLD1 in PC. Thus, the aim of this study was to evaluate the role of PLD1 in PC to find out whether PLD1 could be a useful new target for PC treatment. In order to investigate the role of PLD1 in pancreatic carcinogenesis in vivo, we used a mouse model of early stage PC by conditionally expressing an endogenous KrasLSL-G12D allele in developing pancreatic tissues through the use of pancreas-specific Cre recombinase alleles [KrasLSL-G12D; Ptf1aCre/+ ("KC")] mice contained ductal lesions that mirrored human PanINs, and stochastically developed metastatic PC. We crossed these mice to the Pld1-/- mice and generated the KC; Pld1-/- mice. Cohorts of KC and KC; Pld1-/- mice were euthanized at 4 and we are 8 months old. (6 mice per group at 4 months and 4 mice per group at 8 months. We have now completed enrolling mice. At early age (4 month-old) there was no significant difference in the pancreas weight between the KC mice and the KC;Pld1-/- mice. Interestingly at 8 month old, there was a significant difference (p<0.05) in the pancreas weight between the groups. The pancreas weight of KC mice (mean ± SEM) at 8 months of age was 0.78 ± 0.12 g. In contrast KC;Pld1-/- mice pancreas weight was 0.41 ± 0.08 g, a 47% reduction, when compared to KC mice. Moreover, I am training two students in this project. In particular, the students are evaluating key molecular targets that are affected to elucidate how PLD1 affects pancreatic cancer growth. For this purpose, we evaluated how Pld1 affected the levels of proteins related to the process of cancer progression, using proteomics, immunohistochemistry and western blot. We observed that Pld1 affected the expression levels of proteins related with cell proliferation, cell cycle, inflammation and autophagy. To note, all these biological processes are strongly associated with cancer growth. Some preliminary data are indicating that ERK, and AKT pathways appear strongly modulated by an ablation of PLD1. These pathways are being confirmed in our PC cell and animal models. The outcomes of these studies suggest that PLD1 plays an important role in pancreatic carcinogenesis. Given the importance of PC and the lack of effective treatment options against it, we believe that the proposed work holds the promise of a significant advance in this area. B- Other major aspect of my research interests is the investigation of the anticancer effects of dietary flavonoids. During this period under review we have particularly focused on the beneficial actions of epigallocatechin-3-gallate (EGCG), an abundant polyphenol found in green tea. In this regard: We investigated the capacity of EGCG to suppress the growth of multiple cancer cell in vitro and in vivo, characterizing the underlying mechanisms. We observed that EGCG presents a strong anticancer effect in pancreatic, colon, and lung cancer cells and is a robust combination partner for multiple chemotherapeutics as evidenced by reducing cancer cell growth, in part, by inhibiting the ERK pathway. We also observed that EGCG reduced pancreatic cancer cell growth in a concentration-dependent manner and that EGCG sensitized gemcitabine, a chemotherapeutic drug used in patients, to inhibit pancreatic cancer cell growth in vitro and in vivo. EGCG and gemcitabine, given alone, reduced pancreatic tumor xenograft growth by 40% and 52%, respectively, whereas the EGCG/gemcitabine combination reduced tumor growth by 67%. EGCG enhanced gemcitabine's effect on apoptosis, cell proliferation, cell cycle and further suppressed phosphofructokinase and pyruvate kinase levels. In conclusion, EGCG is a strong combination partner of gemcitabine reducing pancreatic cancer cell growth by suppressing glycolysis. We then investigated the capacity of two dimeric procyanidins composed of epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) isolated from persimmons, to inhibit colorectal cancer (CRC) cell growth and promote apoptosis, characterizing the underlying mechanisms. We observed that ECG and EGCG dimers reduced CRC cell growth by inhibiting EGFR activation at multiple steps, including the disruption of lipid rafts integrity and promoting EGFR degradation. These results shed light on a potential molecular mechanism on how procyanidins-rich diets may lower CRC risk. Results obtained from project A can help raise awareness on the relevance of lipid metabolism and particularly of a lipid-signaling enzyme on cancer growth. Results obtained from project B provide important information about the health benefits of select bioactives.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhu W, Li MC, Wang FR, Mackenzie GG and Oteiza PI. The Inhibitory Effect of ECG and EGCG Dimeric Procyanidins on Colorectal Cancer Cells Growth is Associated with Their Actions at Lipid Rafts and the Inhibition of the Epidermal Growth Factor Receptor Signaling. Biochemical Pharmacology, 2020, May: 175: 113923
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wei R, Hackman RM, Wang Y, Mackenzie GG. Targeting glycolysis with epigallocatechin-3-gallate enhances the efficacy of chemotherapeutics in pancreatic cancer cells and xenografts. Cancers (Basel). 2019 Oct 5;11(10):1496. doi: 10.3390/cancers11101496.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wei R, Wirkus J, Yang Z, Machuca J, Esparza Y, and Mackenzie GG. EGCG sensitizes chemotherapeutic-induced cytotoxicity by targeting the ERK pathway in multiple cancer cell lines. Arch Biochem Biophys. 2020 Oct 15;692:108546. doi: 10.1016/j.abb.2020.108546.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The main outreach of my research has been through the publication of original research articles, as well as seminars and talks on the topic and nutrition and cancer are given at scientific congresses that can reach physicians, other health care providers, public health professionals, and to the general population through general public publications, as well as California cancer patients and their families. Furthermore, the target audience of my educational efforts have been minority students directly benefit from the participation of the summer educational and research experiences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this year, I mentored three Ph.D. graduate students, a visiting graduate student, 1 visiting post-doc and six undergraduate students. I have also led a summer program for two undergraduate minority students. One of the summer students worked directly on the evaluation of the mechanisms of phospholipase d1 in pancreatic carcinogenesis. Other three students worked in the evaluation of EGCG in pancreatic cancer. I have always considered that the experience of an undergraduate student in a research laboratory can be of immense value in their future career decisions, and in their understanding of the scientific thinking. In addition, I have been a member of three dissertation committees, three qualifying committees, and one preliminary exam committee. I also received in my laboratory two international scholars: Dingyuan Luo, a medical doctor from Sun Yat-Sen University (one year research visit Dec 2017-Dec 2018), and Ran Wei, a visiting graduate student from Zhejiang University, who spent 2 years in the lab completing her research work in my laboratory for graduation. Of note, we have published several manuscripts during their visit. These visits have a direct impact on the career advancement of the international visitors. It is noteworthy that they also provide graduate and undergraduate students in my laboratory the possibility of interaction with highly motivated and trained scientists/students, and open their minds to international nutritional concerns that are also of relevance locally. How have the results been disseminated to communities of interest?We have disseminated our research through: a) The presentation of several abstracts/posters to national and international conferences. b) Publication of research papers. c) Teaching undergraduate courses on nutrition and health at UC Davis. d) Supporting the career advancement of minority students. I have been and I am a mentor for a student participating of the UC Davis Continuing Umbrella of Research Experiences (CURE) program, which provides research training for diverse UC Davis undergraduates in their junior and senior years. I am the Director of the NUTGAP program and I am mentor of NSF LSAMP/CAMP program, who recently joined the lab What do you plan to do during the next reporting period to accomplish the goals?I will continue the ongoing projects focused on: A) the understanding the role and mechanism of action of the lipid-signaling enzyme phospholipase D1 (PLD1) in pancreatic cancer growth; and B) investigating the anticancer effects of phytochemicals present in US agricultural products in certain cancers.

Impacts
What was accomplished under these goals? The long-term goal of my research group is to elucidate the effect that diet and select dietary components have on cancer progression. During the current period we have worked on the following two projects: A- One major aspect of my research interest is to define the role of the lipid-signaling enzyme phospholipase D1 (PLD1) in pancreatic cancer growth. We hypothesize that PLD1 is a key enzyme in PDA growth. This is a work in progress. We will employ biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance PLD1 as a novel therapeutic target. Pancreatic cancer (PC) is a deadly cancer with unsatisfactory treatment options. The poor prognosis of PC requests the exploration of new ways to treat this disease. Phospholipase D1 (PLD1) is a lipid-signaling enzyme that has been shown to play a role in the progression of various types of cancer. However, there is no information about the role of PLD1 in PC. Thus, the aim of this study was to evaluate the role of PLD1 in PC to find out whether PLD1 could be a useful new target for PC treatment. In order to investigate the role of PLD1 in pancreatic carcinogenesis in vivo, we used a mouse model of early stage PC by conditionally expressing an endogenous KrasLSL-G12D allele in developing pancreatic tissues through the use of pancreas-specific Cre recombinase alleles KrasLSL-G12D; Ptf1aCre/+ ("KC") mice contained ductal lesions that mirrored human PanINs, and stochastically developed metastatic PC. We crossed these mice to the Pld1-/- mice and generated the KC; Pld1-/- mice. Cohorts of KC and KC; Pld1-/- mice were euthanized at 4 and we are 8 months old. (6 mice per group at 4 months and 4 mice per group at 8 months. We are currently enrolling additional mice. At early age (4 month-old) there was no significant difference between the KC mice and the KC;Pld1-/- mice. However, at 8 month old, there was a significant difference (p<0.05) in the pancreas weight at sacrifice. The pancreas weight of KC mice (mean ± SEM) at 8 months of age was 0.78 ± 0.12 g. In contrast KC;Pld1-/- mice pancreas weight was 0.41 ± 0.08 g which was significant lower than KC mice We also pursued to study how PLD1 affects pancreatic cancer growth. For this purpose, we evaluated how Pld1 affected the levels of proteins related to the process of cancer progression, using proteomics. We observed that Pld1 affected the expression levels of proteins related with cell proliferation, cell cycle, inflammation and autophagy. To note, all these biological processes are strongly associated with cancer growth. Currently, key proteins of these pathways are being confirmed in our PC cell and animal models. The outcomes of our initial studies suggest that PLD1 plays an important role in pancreatic carcinogenesis. Given the importance of PC and the lack of effective treatment options against it, we believe that the proposed work holds the promise of a significant advance in this area. B- Other major aspect of my research interests is the investigation of the anticancer effects of dietary flavonoids. During this initial period under review we have particularly focused on the beneficial actions of epigallocatechin-3-gallate (EGCG), an abundant polyphenol found in green tea. In this regard: B.1. We investigated the capacity of EGCG to suppress pancreatic cancer cell growth in vitro and in vivo, characterizing the underlying mechanisms, focusing on the effect of EGCG on glucose metabolism. EGCG reduced pancreatic cancer cell growth in a concentration-dependent manner and the growth inhibition effect was further enhanced under glucose deprivation conditions. Mechanistically, EGCG induced ROS levels concentration-dependently. EGCG affected glycolysis by suppressing the extracellular acidification rate through the reduction of the activity and levels of the glycolytic enzymes phosphofructokinase and pyruvate kinase. Cotreatment with catalase abrogated EGCG's effect on phosphofructokinase and pyruvate kinase. Furthermore, EGCG sensitized gemcitabine to inhibit pancreatic cancer cell growth in vitro and in vivo. EGCG and gemcitabine, given alone, reduced pancreatic tumor xenograft growth by 40% and 52%, respectively, whereas the EGCG/gemcitabine combination reduced tumor growth by 67%. EGCG enhanced gemcitabine's effect on apoptosis, cell proliferation, cell cycle and further suppressed phosphofructokinase and pyruvate kinase levels. In conclusion, EGCG is a strong combination partner of gemcitabine reducing pancreatic cancer cell growth by suppressing glycolysis. We have published this work (see above). We are currently working on evaluating how EGCG affects cell senescence and in various cancer models, to see if it is a beneficial adjuvant drug. Results obtained from project A can help raise awareness on the relevance of lipid metabolism and particularly of a lipid-signaling enzyme on cancer growth. Results obtained from project B provide important information about the health benefits of select bioactives.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wei R, Hackman RM, Wang Y and Mackenzie GG. Targeting Glycolysis With Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts. Cancers (Basel). 2019 Oct 5;11(10). pii: E1496. doi: 10.3390/cancers11101496..
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wei R, Penso NEC, Hackman RM, Wang Y, Mackenzie GG. Epigallocatechin-3-Gallate (EGCG) Suppresses Pancreatic Cancer Cell Growth, Invasion, and Migration partly through the Inhibition of Akt Pathway and Epithelial-Mesenchymal Transition: Enhanced Efficacy when Combined with Gemcitabine. Nutrients. 2019 Aug 9;11(8). pii: E1856. doi: 10.3390/nu11081856.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:During this first year, the main outreach of my research has been through the publication of an original research article that can reach physicians, other health care providers, public health professionals, and to the general population through general public publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this initial year, I mentored two Ph.D. graduate student, a visiting graduate student, and 1 visiting post-doc. I was member of one dissertation committees, three qualifying committees, and multiple preliminary exam committee, and trained 3 undergraduate students. I have always considered that the experience of an undergraduate student in a research laboratory can be of immense value in their future career decisions, and in their understanding of the scientific thinking. Furthermore, they understand the relevance of research and of its short- and long-term translation for public health. I also received in my laboratory several international scholars: Dingyuan Luo, a medical doctor from Sun Yat-Sen University (one year research visit 2017-2018), and Ran Wei, a visiting graduate student from Zhejiang University, who is completing her research work in my laboratory for graduation. These visits have a direct impact on the career advancement of the international visitors. It is noteworthy that they also provide graduate and undergraduate students in my laboratory the possibility of interaction with highly motivated and trained scientists/students, and open their minds to international nutritional concerns that are also of relevance locally. How have the results been disseminated to communities of interest?We have disseminated our research through: a) The presentation of several abstracts/posters to national and international conferences. b) Publication of research papers. c) Teaching undergraduate courses on nutrition and health at UC Davis. d) Supporting the career advancement of minority UCD students. I have been and I am a mentor for a student participating of the UC Davis Continuing Umbrella of Research Experiences (CURE) program, which provides research training for diverse UC Davis undergraduates in their junior and senior years. What do you plan to do during the next reporting period to accomplish the goals?I will continue the ongoing projects focused on: A) the understanding the role of the lipid-signaling enzyme phospholipase D1 (PLD1) in pancreatic cancer growth; and B) investigating the anticancer effects of phytochemicals present in US agricultural products in certain cancers.

Impacts
What was accomplished under these goals? The long-term goal of my research group is to elucidate the effect that diet and select dietary components have on cancer progression. During the current period we have worked on the following two projects: A) One major aspect of my research interest is to define the role of the lipid-signaling enzyme phospholipase D1 (PLD1) in pancreatic cancer growth. We hypothesize that PLD1 is a key enzyme in PDA growth. This is a work in progress. We will employ biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance PLD1 as a novel therapeutic target. During this initial period under review, we have worked toward developing pancreatic cancer cells in which PLD1 has been deleted, using CRISPR/Cas technology. We were successful in generating PLD1 knockdown (KD) pancreatic cancer cells using CRISPR/Cas technology. We already established the MIA PaCa-2 PLD1 KD cells. We are currently working on using this technology to generate the human Panc-1 PLD1 KD cells and the mouse KPC PLD1 KD cells. These cells will allow us to directly evaluate the contributing of this lipid-signaling enzyme on pancreatic cancer growth. Furthermore, In order to investigate the role of PLD1 in pancreatic carcinogenesis in vivo, we used a mouse model of early stage pancreatic cancer by conditionally expressing an endogenous KrasLSL-G12D allele in developing pancreatic tissues through the use of pancreas-specific Cre recombinase alleles. KrasLSL-G12D; Ptf1aCre/+ ("KC") mice contained ductal lesions that mirrored human PanINs, and stochastically developed metastatic PDA. We crossed these mice to the Pld1-/- mice and generated the KC; Pld1-/- mice. Cohorts of KC and KC; Pld1-/- mice were euthanized at 4 and we are 8 months old. (6 mice per group at 4 months and 4 mice per group at 8 months. We are currently enrolling additional mice. Initial evidence suggests that ablation of PLD1 affects pancreatic cancer growth. This study is ongoing. B) Another major aspect of my research interests is the investigation of the anticancer effects of dietary flavonoids. During this initial period under review we have particularly focused on the beneficial actions of epigallocatechin-3-gallate (EGCG), an abundant polyphenol found in green tea. In this regard: B.1. We investigated the capacity of EGCG to suppress breast cancer cell growth in vitro and in vivo, characterizing the underlying mechanisms, focusing on the effect of EGCG on glucose metabolism. EGCG reduced breast cancer cell growth in a concentration- and time-dependent manner, induced apoptotic cell death and affected autophagy. Mechanistically, we observed that EGCG significantly inhibited the activities and mRNA levels of the glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), and lactic dehydrogenase (LDH), critical players in regulating glycolysis and key players of EGCG mechanism of action. In vivo, EGCG reduced breast tumor growth in a dose-dependent manner, reduced glucose and lactic acid. In conclusion, EGCG exerts anti-tumor effect through the inhibition of key enzymes that participate in the glycolytic pathway and the suppression of glucose metabolism. We have published this work (see above). We are currently working on evaluating the role of EGCG in pancreatic cancer prevention and treatment. Results obtained from project A can help raise awareness on the relevance of lipid metabolism and particularly of a lipid-signaling enzyme on cancer growth. Results obtained from project B provide important information about the health benefits of select bioactives.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wei R, Mao L, Xu P, Zheng X, Hackman RM, Mackenzie GG and Wang Y. Suppressing glucose metabolism with epigallocatechin-3-gallate (EGCG) reduces breast cancer cell growth in preclinical models. Food & Function, 2018; Nov 14;9(11):5682-5696. doi: 10.1039/c8fo01397g.