Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Human Nutrition & Foods
Non Technical Summary
The obesity epidemic is a major concern for communities in all states. An important factor for disease risk is the location where the fat is accumulating in the body. Especially the abdominal fat depots -apple-shaped body- contribute to coronary heart disease, hypertension, stroke, diabetes, and atherosclerosis but also to chronic kidney and liver disease, tissue fibrosis, and other diseases even in people with normal body weight. Obesity increases inflammatory markers but the molecular mechanisms of how abdominal adipose tissues increase the risk of these diseases are not fully understood. Obesity also increases the risk of developing several cancers such as prostate, breast, colon, liver, and ovarian cancer and significantly increases ovarian cancer mortality. The risk of ovarian cancer is especially high when the onset of obesity occurs early in life and, similar to other obesity-related diseases when the excess adipose tissue is abdominal. There is a distinct need for identifying the underlying mechanisms of how obesity and different fat depots contribute to the development of diseases. Here we are proposing to determine the molecular mechanisms of how obesity affects the growth of the early and late stages of ovarian cancer. We will investigate how obesity changes the composition of adipose tissues and how these changes will affect cancer development, disease progression, and, finally, metastasis to secondary sites. Since ovarian cancer has one of the highest incidence-to-death ratios of all cancers, understanding how obesity affects the dynamics of the interactions of cancer cells with their environment are critical steps for the development of potential therapeutic targets, and the molecular controls for novel strategies to prevent or suppress ovarian cancer in high-risk groups While we are focusing here on ovarian cancer, the determination of the molecular mechanisms will also support the risk assessment of other diseases in risk groups before the onset of disease. This project will also provide the scientific basis for evidence-based recommendations or guidelines for healthy food intake to maintain a healthy weight or the prevention of obesity to improve preventive and treatment strategies, and, subsequently, prevent obesity-mediated diseases. Childhood obesity in Virginia is currently at 15% in the 2-4-year-old age group which is among the highest in the country (12th most obese state). The obesity rate has been constantly increasing over the last decade and has reached 30% in the 26-44-year-old Virginians (https://stateofchildhoodobesity.org/states/va/). Race, socio-economic status, and lack of insurance/access to standard treatment contribute to the incidence of obesity (childhealthdata.org) and to the higher mortality of ovarian cancer[10]. In Virginia alone, 1,825 per 100,000 women die of ovarian cancer per year compared to its incidence (2,629 per 100,000)[11]which is comparable to other states. The lack of specific symptoms reduces the early detection rate and diagnosis is often delayed until late disease stages when cancer has spread throughout the abdomen and causes pain. Obesity causes unspecific symptoms such as pelvic pressure, fatigue, or indigestion[12] that may mask the same symptoms caused by ovarian cancer, delaying diagnosis, and thereby increasing the mortality rate. Thus, obesity will increase the cancer risk disproportionally in poor black women with a high rate of obesity (>38%) over the next decades. While weight-loss strategies are in place, they often are not successful. Long-term intervention and prevention strategies that can improve by the molecular markers developed in this project are necessary to suppress or delay ovarian cancer or prevent the development of other obesity-related diseases.In the US, 17.9% of the GDP is spent on healthcare (CDC website). Obesity raises health care costs by about 30% and contributes significantly to lost productivity, lost wages due to illness and disability, and loss of quality of life. Therefore, preventing obesity-related comorbidities such as cancer will save lives and will lessen the economic burden for taxpayers and employers.We expect to find that obesity will expand the SVF in all adipose tissues but will change their composition only in the abdominal tissues but not in the subcutaneous tissues. We expect that the cancer cells adapt to the conditions in the peritoneal cavity by reducing their metabolism and that only the SVF derived from the abdominal adipose tissues can support these adaptations. We anticipate that the inhibition of metabolic adaptation will slow the metastatic processes and increase the survival of mice bearing ovarian cancer metastases. Understanding the dynamics of the pre-metastatic tumor microenvironment as well as characterizing the cellular and molecular changes involved in the generation of a permissive niche for ovarian metastases are critical steps for elucidating potential therapeutic targets and developing novel strategies to prevent or suppress ovarian cancer metastasis in high-risk groups. The results from these studies will provide a mechanistic link between abdominal obesity and the increased ovarian cancer risk. Since aggregation also enhances the metastatic potential of breast and colon cancer cells, the results from the proposed studies can be translated into the suppression of other metastatic diseases. Furthermore, we expect that the identified SVF changes may also be relevant for other obesity-mediated diseases.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We will focus first on how obesity changes the composition of visceral and subcutaneous adipose tissue since visceral fat confers a higher disease risk while subcutaneous fat may be protective and has been used in many studies as inert or non-reactive control. We will then use in vitro experiments to determine how the SVF from the different adipose tissues affect the cancer cells, focusing on the adaptation of the cellular metabolism. Our central hypothesis is that the capacity of ovarian cancer cells to adapt to an environment low in oxygen and nutrient levels during dissemination but is changed after adhesion and invasion at metastatic sites is critical for their survival. Here we hypothesize that obesity will not only provide alternative resources for cancer cells to survive the hostile peritoneal environment but also aid in their ability to adjust their cellular functions to the conditions in their microenvironment. We will use our established mouse cell system that represents all stages of ovarian cancer in addition to novel human immortalized and transformed ovarian and fallopian tube cell lines to determine the molecular mechanisms of how the SVF cells support the cancer cells' adaptation to their cultural environment. Lastly, we will determine if we can suppress metastasis by inhibiting these critical signaling events using commercially available inhibitors of the identified targets. We will use culture techniques that represent conditions of the specific disease stage, beginning with adherent cultures that represent the primary tumor to 3D cultures for disseminating cancer aggregates and finally 3D invasion models with the appropriate levels of oxygen and nutrients that are found at specific sites. Thus, our studies also allow for the identification of molecular events that increase the risk of developing cancer in obese women. Our preliminary data indicate that the modulation of cellular energetics allows for the adaptation of cancer cells to the dynamic tumor environment that may be supported by recruitment of the SVF from the abdominal adipose tissues and suggest that the SVF-induced increase in metabolic plasticity is a potential mechanism of how obesity increases ovarian cancer. Our overall hypothesis is that ovarian cancer adapts both mitochondrial morphology and function to the availability of oxygen, nutrients, and adhesion. We further hypothesize that the development of the "thrifty" phenotype as well as the reversal to a fully functional phenotype is supported by obesity.Our overall goal is to identify specific signaling events that allow for the adaptation of cancer cells to a changing environment and to understand how obesity contributes to this adaptation. If these signaling events are druggable targets, this could lead to the development of a prevention strategy to reduce the ovarian cancer risk and potentially the risk of other diseases in obese women. We propose the following objectives:Objective 1: Determine how obesity affects the composition of disseminating ovarian cancer aggregates and the ascites in women with ovarian cancer We have previously shown that feeding an HFD in mice leads to a change in SVF composition and in the expression of genes that generate an inflammatory environment and can support cancer cell survival. Here we will test the hypothesis that obesity changes the SVF in abdominal adipose tissues and thereby the composition of disseminating cancer aggregates. Together with changes in the ascites secretome, these changes contribute to the survival of ovarian cancer cells. We will collect adipose tissues (mesenteric, omental, ovarian, subcutaneous) and ascites from women undergoing surgery in collaboration with Carilion clinic (IRB 19-642) and isolate the SVF. We will characterize the composition of the SVF in the adipose tissues and the cellular fraction of the ascites and use qRT-PCR arrays to assess gene expression levels as a function of waist circumference. The ascites plays an important role in transporting and seeding cancer cells along with their flow pattern and is critical for their survival. Mass spectrometry will be used to assess how key bioactive lipids, growth factors, and nutrients are changed in correlation to waist circumference, and in vitro assays are used to determine the impact of ascites on cancer cell growth, motility, and invasion.Objective 2: Characterize the changes in cellular energetics during ovarian cancer development, progression, and metastasis.Exfoliated ovarian cancer cells need to adapt to low oxygen and nutrient levels in the ascites to survive. After adhesion to the OFB or other peritoneal sites, the cells need to upregulate their energy production to meet the demand for migration, invasion, and outgrowth. We hypothesize that aggregates reduce cellular respiration in non-permissive conditions but up-regulate metabolism upon adherence to metastatic sites. We further hypothesize that the recruited SVF from the adipose tissue of obese women enhances both metabolic downregulations during aggregation and the upregulation of metabolism after adhesion. We will investigate this phenotypical switch by simulating peritoneal metastasis in non-permissive conditions followed by adhesion and outgrowth with a switch to optimal growth conditions. We will focus on changes in mitochondrial morphology and function because, despite the importance of the highly glycolytic metabolism of rapidly growing cancer cells, the central role of metabolism in the adaptation of cancer cells to their dynamic microenvironment is still unknown. Here we will characterize the phenotypical switch of the cells during the metastatic process from dormancy to proliferation as it relates to mitochondrial morphology and function.Objective 3: Determine how the inhibition of mitochondrial biogenesis affects ovarian cancer metastatic properties.The studies under objective 1 will identify signaling events that contribute to the adaptive phenotype of successful ovarian metastases that will be targeted in studies under this objective. We will begin our studies with the antimicrobial agent tigecycline that has been shown to reduce mitobiogenesis via inhibition of mitochondrial translation; this suppressed tumor growth in leukemia, triple-negative breast, and ovarian cancer. Tigecycline inhibits proliferation by arresting ovarian cancer cells in G2/M phase while also inhibiting mitochondrial translation and respiration. Specifically, tigecycline inhibits mitochondrial ribosomal translation of key respiratory complexes, preventing oxidative phosphorylation, and leading to mitochondrial dysfunction, oxidative stress accumulation, activation of AMPK, inhibition of mTOR signaling, and suppression of mitochondrial respiration. Here we will use tigecycline to prevent mitochondrial biogenesis stimulated by adhesion in order to provide energy for proliferation. Specific PGC1a, cMyc, glucose transporter or other important signaling molecules in addition to inhibitors of specific ETC complexes will be used to confirm the importance of mitobiogenesis and up-regulation of cellular bioenergetics for a successful metastatic outgrowth. Using the strategy outlined above, we will confirm that these inhibitors suppress mitobiogenesis and upregulation of respiration as indicated by ATP synthesis. We will then use in vitro assays to correlate these changes to the altered metastatic capacity, quantifying functional events such as adhesion, invasion, and proliferation under the conditions that reflect dissemination (spheroids in ultra-low adhesion plates, hypoxia, low glucose) or secondary outgrowth (outgrowth in collagen, adhesion to and clearance of mesothelial layers, invasion of the underlying matrix, increasing levels of oxygen and glucose). To confirm a reduced metastatic capacity, cells treated with the inhibitors will be injected i.p. into syngeneic C57/BL6 mice, and the tumor burden will be determined.
Project Methods
Methods for objective 1Discard tissues will be collected from women with ovarian cancer: ascites, subcutaneous WAT, OFB, mesenteric WAT, and ovarian depots. The ascites is centrifuged, the supernatant frozen in aliquots and used as malignant ascites in in vitro studies determining metabolism, proliferation, migration, and invasion using assays described above and correlated to the waist circumference of the patient (normal, overweight, obese). This will identify cellular functions that are affected by the obese ascites and shed light on how ascites can support the survival of disseminating ovarian cancer cells. The SVF is isolated from the collected tissues and characterized using flow cytometry or will be placed into RNAlater for qPCR analyses as described. Information obtained from patients includes weight, height, waist circumference, type and stage of ovarian cancer, and treatment. This will allow for the correlation of data to abdominal adiposity and thereby to ovarian cancer riskMethods for objective 2 and 3Cell lines: We will use our syngeneic mouse ovarian surface epithelial (MOSE) cell model representing early-benign (MOSE-E), slow-developing (MOSE-L), and fast-developing (MOSE-LTICv) serous ovarian cancer. MOSE-E does not form viable spheroids and serves as adherent controls. FNLE, a fallopian tube-like human cancer cell line, and SOV3, a human serous ovarian cancer cell line will be used as a comparison to human serous ovarian cancerPrimary tissue: Peritoneal lavage will be performed on obese female C57BL/6 mice with 5ml of sterile PBS; the recovered fluid is centrifuged, combined from all mice, aliquoted, and frozen until use as obese lavage. SVF cells are isolated from the abdominal fat pads as described[54]. SVF fraction from the subcutaneous fat serves as a control. Cells are grown for 3 days, combined from all mice, and used for experiments or frozen. The human SVF collected under objective 1 will be processed as the murine tissues and used in studies involving the human cells.Culture conditionsHypoxia: Oxygen levels of about 10% in the peritoneal cavity of healthy people and as low as 1% in malignant ascites were reported. Nutrient deprivation: Mimicking ascites, cells are grown in serum-free DMEM with 5mM glucose, 0.3, and 0.5% charcoal-stripped FCS that supports slow proliferation. 25mM glucose and FCS will be added when mimicking primary tumor or metastasis after angiogenesis. 3D peritoneal cell culture models: Single spheroids (defined number and composition) are generated in ultra-low adherence plates (96wells, round bottom). For respiration studies, multiple spheroids of similar size are generated in ultra-low adherence 6-well plates. These spheroids represent disseminating aggregates. 3D Outgrowth is determined after embedding spheroids in collagen via imaging with a Nikon 80i epifluorescence microscope with the NIS Elements software. Adherence and invasion is quantitated by seeding mesothelial cells (Celprogen) onto a thick collagen plug; after grown to confluence the spheroids will be seeded onto the monolayer and adhesion will be determined after 3h and gentle washes to discard non-adherend spheroids by light microscopy; invasion will be monitored with the confocal Leica SP8 DMi8 microscope, equipped with a cell culture stage with oxygen regulation. Images are processed with the Leica LASX software. Adherent cells serve as controls.The combination of these culture conditions is used to identify how the cells adapt to their dynamic microenvironment with the following measurements:Proliferation is determined by alamar blue assay as described. This is complemented with measurements of spheroid diameter.Metabolism: The following parameters are determined to understand how cells adapt to the conditions in their microenvironment to support survival and growth: glucose uptake, lactate secretion, metabolic flexibility, and substrate preference (extended to glutamine, pyruvate and lactate), and TCA flux as described. Next, glucose and fatty acid oxidation and fatty acid and cholesterol synthesis are assessed. The metabolic core at VT will assist with these assays allowing for fast analysis of conditions and treatment combinations.ATP production, proton leak, maximal respiration, and spare respiratory capacity, ECAR) is measured with the Seahorse Extracellular Flux Analyzer in a 96well format for a fast screen of the responses to culture conditions. We have adapted the protocols to allow for tracing the lower respiration of the spheroids. Mitochondria: We then will investigate how these changes in cellular metabolism rely on mitochondria number and shape. Morphology is determined by confocal and super-resolution microscopy in spheroids grown in the combination of variables described above. The spheroids are fixed in paraformaldehyde or methanol, and mitochondria will be visualized with an TOMM20 antibody (integral mitochondria protein) or CF568 (Biotium) (specific for STORM imaging). In conditions that change the mitochondrial number, we will investigate changes in the balance of mitobiogenesis and mitophagy by determination of key regulators of mitobiogenesis (PGC1a, TFAM, TOMM20) and mitophagy (BNIP3, NIXL, LC3B) by Western blotting. Mitochondrial turnover is assessed by transfecting cells with MitoTIMER (plasmid from Addgene) and live-imaging with a Zeiss LSM 880 confocal microscope.Ascites control: Our data show that obese ascites support cancer cell survival (Fig.1). A comprehensive analysis of the malignant or obese secretome is not available. Here we will add 5% obese lavage to serum-free medium as obesity controls. In objective 3 we will optimize the working concentration of tigecycline using an MTT assay to prevent treatment cytotoxicity and confirm that the inhibitor prevents mitochondrial translation through western blotting (TOMM20). We then will determine if the inhibition of mitobiogenesis suppresses the phenotypical switch by assessing respiration, metabolism, proliferation, adhesion, and invasion as described above. To confirm that this inhibition is suppressing metastasis in vivo, female C57BL/6 will be injected with MOSE-LTICv and subsequently treated with continuous subcutaneous injections with tigecycline or PBS (controls) after 5 days. Alternatively, the cancer cells will be treated with tigecycline before injection. Data Analyses: All in vitro experiments include controls for the variables of interest (oxygen, glucose levels, adherent or vehicle controls) and are performed in triplicate in 3 biological replicates. The use of defined spheroids in vitro (exact cell number, composition) limits the influence of variables (size, hypoxic core, viability) that can affect gene expression and function. The characterization of adipose tissues and ascites is performed in individual samples and correlated to waist circumference. Flow cytometric analyses are quantitated with Flowjo software; qRT-PCR experiments will be run in triplicates and quantitated with the ABI software, normalized to the housekeeping gene L19 since we have detected changes in β-actin and GAPDH during MOSE progression.Parametric data will be analyzed using ANOVA followed by Scheffe's multiple comparison method. The main effect of obesity, culture conditions, and cell functions will be examined as a factorial arrangement. Nonparametric data will be analyzed by using the Mann-Whitney's U test followed by a Dunn's multiple comparisons test. ANOVA will be performed by using the general linear model procedure of SAS. Similar procedures will be used to examine the association of culture variables and function population. The Laboratory for Interdisciplinary Statistical Analysis at Virginia Tech will assist with these calculations and the more complex calculations as needed.