Abstract 2934: GSK3β modulates chemoresistance in epithelial ovarian cancer

Introduction: Epithelial ovarian cancer is one of the most common gynecological malignancies and the fifth most frequent cause of cancer death in women, affecting over 22,000 women annually. Nearly 15,500 affected women die from this disease annually, and chemoresistance from the commonly prescribed platinum-based drug, carboplatin, is a major contributor to this mortality. Previous studies have identified genes with CpG islands that are methylated and transcriptionally silenced in resistant epithelial ovarian cancer patients. One of these genes is GSK3β, an important regulator of apoptosis and cell growth in the Wnt pathway. Thus, understanding the role of GSK3β suppression in chemoresistance of epithelial ovarian cancer can help contribute to more effective treatments for this disease. By performing different assays our study examined the functional role that GSK3β plays in carboplatin chemoresistance. Procedure: Human ovarian surface epithelium (HOSE) 6-3 cell line was utilized, which is characterized as sensitive to carboplatin therapy. The cells were studied in six groups: 1) untreated; 2) treated with Lithium Chloride (LiCl); 3) treated with carboplatin; 4) treated with carboplatin and LiCl; 5) treated with doxorubicin as control; 6) treated with doxorubicin and LiCl as another control. LiCl is known to suppress GSK3β gene expression. We took images of cells using a fluorescence microscope. We also performed the Neutral Red Dye assay that determines cell viability, Vybrant® MTT Cell Assay which measures amount of non-viable cells, Caspase 3 Assay which measures cell apoptosis, and we did cell counting using a hemocytometer and a light microscope. Data analysis was done by T-tests using Microsoft Excel®, with p \u3c .05 for significance. Summary of Data: More growth was observed in the carboplatin and LiCl group compared to the carboplatin group alone on microscopy. Neutral Red Dye assay: Compared to the cells exposed to carboplatin alone, those exposed to carboplatin and LiCl were more viable (p \u3c0.01). Vybrant® MTT Cell Assay: the cells treated with carboplatin and LiCl showed lesser amounts of nonviable cells as compared to the carboplatin alone group (p\u3c0.01). Caspase 3 Assay: the cells treated with carboplatin and LiCl were less apoptotic, compared to cells treated with carboplatin alone (p \u3c0.01). Cell Counting: cells treated with carboplatin and LiCl had significantly more growth compared to the cells treated with carboplatin alone (p \u3c0.01). Conclusion: Our results show that cells with suppressed GSK3β had increased proliferation and reduced apoptosis, strongly suggesting that silenced GSK3β expression contributes to carboplatin resistance and GSK3β expression is vital to carboplatin chemosensitivity. Future in vivo studies could further investigate the role of GSK3β methylation to facilitate the design of potential genome-guided treatments for patients with chemoresistant epithelial ovarian cancer

Changes in Breast Cancer Care in New York During the COVID-19 Pandemic

Breast cancer is the second most common malignancy among women in the United States. As such, the COVID-19 pandemic has caused medical facilities to change their methods of operation since March of 2020, including changes in diagnosis and treatment plans. New York (NY) has an unusually high incidence of breast cancer. This study analyzed the effects of the COVID-19 pandemic on breast cancer care (BCC) in NY. Women in NY that were diagnosed with or in remission for breast cancer were asked to take an online, anonymous survey regarding their BCC experience. For patients in treatment, 26% of women wished they had greater emotional support or had a family member included in their appointments. 39% of women do not feel they are receiving as good of care as before, while 31% feel they are receiving the same level of care. Additionally, 41% of women feel they received the same level of care over telemedicine. Our data show a negative correlation between the quality of care received during the pandemic and the wish for more emotional support and inclusion of supportive members in the care process (nonsignificant). There was less of a negative correlation between the quality of telemedicine care received during the pandemic and the wish for more emotional support and inclusion of supportive members in the care process (nonsignificant). This indicates that most women lacking emotional support reported worse BCC experiences, and telemedicine use was not as troublesome to patients as the lack of emotional support. Most women in treatment and in remission reported negative feelings like fear when asked about their BCC experiences. Our data show the importance of emotional support for breast cancer patients and those in remission during the COVID-19 pandemic. Our work could also provide clinicians with the knowledge necessary on how breast cancer care should be handled in an evolving pandemic such as COVID-19

Inhibitions of GSK3β Modulates Cell Death in Epithelial Ovarian Cancer

Epithelial ovarian cancer is one of the most common gynecological malignancies and the fifth most frequent cause of cancer death in women, affecting over 22,000 women annually. Nearly 15,500 affected women die from this disease annually, and chemoresistance from the commonly prescribed platinum-based drug, carboplatin, is a major contributor to this mortality rate. Previous studies have identified genes with CpG islands that are methylated and transcriptionally silenced in resistant epithelial ovarian cancer patients. One of these genes is GSK3β, an important regulator of apoptosis and cell growth in the Wnt pathway. Thus, understanding the role of GSK3β suppression in chemoresistance of epithelial ovarian cancer can help contribute to more effective treatments for this disease. By performing assays of cell growth, viability, and apoptosis, our study examined the functional role that GSK3β plays in carboplatin mediated apoptosis. Our results suggest that cells with suppressed GSK3β had increased proliferation and reduced apoptosis. We conclude that silenced GSK3β expression might therefore contribute to carboplatin resistance seen in tumors and our in vitro analysis suggests that GSK3β expression is vital to carboplatin chemosensitivity. Future research is required to further investigate the role of GSK3β methylation to facilitate the design of potential genome-guided treatments for patients with chemoresistant epithelial ovarian cancer

Abstract 2421: The role of p53 mutational status and SOX9 suppression in chemotherapy response of ovarian cancer

Ovarian cancers are highly heterogeneous where platinum based chemotherapy which induces DNA crosslinking resulting in apoptosis of the cell is the preferred treatment. However, many patients are intrinsically resistant or quickly develop resistance. The Sox factors are a large family of transcription factors that play important roles in tumor development and progression in a variety of human malignancies and diverse developmental processes, but their impact in clinical tumorigenesis is still unclear. An analysis of genomic changes in ovarian cancer has provided the most comprehensive and integrated view of cancer genes for any cancer type to date. Ovarian serous adenocarcinoma tumors from 500 patients were examined by The Cancer Genome Atlas (TCGA) Research Network and analyses are reported in a recent issue of Nature. This evidence suggests that epigenetic deregulation, such as methylation, may be a key factor in the onset and maintenance of chemoresistance. Previous microarray analysis results in our lab correctly identified a subset of about 300 genes that when methylated altered the chemoresistance of the ovarian epithelium cells in culture. Of the genes identified in the analysis we further set out to characterize oncogenes and tumor suppressor genes that interact with the guardian of the genome, TP53, to determine if we could elucidate the mechanism by which it increased resistance. Using several in-vitro assays, we determined that the loss of p53 in conjunction with SOX9 decreased the level of apoptosis in response to carboplatin. Furthermore, in cells with mutated p53/SOX9 show an increase in tumorigenesis. Regulation of several pathways with p53 mutations in ovarian cancer might represent a therapy response prediction and could be a future therapeutic target for ovarian cancer. In addition, the crosstalk between p53/SOX9 and epigenetic regulators may present a valid treatment option for increasing carboplatin sensitivity in resistant patients

Strenuous Exercise Increases the Risk of Oxidative Stress in Ironman Triathlon Participants

Regular physical activity has been linked to greater overall health. Literature review and studies have also defined regular physical activity as a reducer of life-threatening illnesses such as cardiovascular disease, diabetes and obesity. However, long increments of strenuous exercise can produce oxidative stress and muscle fatigue in the human body. The increase in oxygen consumption during strenuous exercise leads to elevated reactive oxygen species (ROS). Cells continuously produce free radicals and reactive oxygen species as part of metabolic processes in the body. These free radicals are neutralized by an antioxidant defense system in the body consisting of enzymes, such as catalase, and non-enzymatic antioxidants. An Ironman Triathlon consists of a 2.4-mile (3.86 km) swim, a 112-mile (180.25 km) bicycle ride and a marathon 26.2-mile (42.2 km) run, raced in that order and without a break. It is widely considered by athletes to be one of the most demanding sporting events in the world. It is hypothesized that a physically challenging event such as the Ironman Triathlon can be linked to elevated cortisol levels, increased occurrence of DNA damage, elevated concentrations of ROS, and consequently increased oxidative stress in humans. In order to derive conclusive results regarding the hypothesis, groups containing athletes who completed the full Ironman race, the half Ironman race, and a control group of moderately active individuals were established and individuals were required to report Garmin Smartwatch health and wellness data. The half Ironman consists of a 1.2-mile (1.93 km) swim, a 56-mile (90.12 km) bicycle ride and a marathon 13.1-mile (21.1 km) run, raced in that order and without a break. Several protocols were then applied to derive data necessary to complete the research. After the participants were selected, their saliva was collected in a non-invasive fashion and was used in the Elisa Saliva Kit to determine cortisol concentration. The saliva samples were also utilized to perform DNA and RNA extraction; and the resulting products were analyzed for quantity and quality of the DNA and RNA. Real time PCR allows scientists to monitor PCR while it is occuring. In this technique, luminescence is produced by reporter molecules as the PCR products increase with every cycle. To determine ROS concentration, the ROS-Glo assay, which provides a light signal that is proportional to the ROS in a given sample, was utilized. An additional marker of oxidative stress is 8-oxo-2-deoxyguanosine(8-oxo-dG). The OxiSelect™ Oxidative DNA Damage ELISA uses antibody and antigen interactions to report the concentration of 8-oxo-dG in a sample. Furthermore, the results indicate an increase in enzymatic indicators of elevated ROS, elevated cortisol levels, and disruption of sleep in the participating athletes after the race. In conclusion, the athletes who completed the full Ironman triathlon experienced increased amounts of oxidative stress than their less active counterparts in the control group, as was denoted by the elevated cortisol levels, increased 8-oxo-dG concentrations, and increased ROS concentrations.Such a rigorous event negatively impacted participants and caused oxidative stress.Faculty Sponsor: Noelle Cutte

Abstract 4525: Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor that when induced regulates the expression of many genes involved in cytoprotective stimuli, which attenuates apoptosis and improves survival. Increased expression of HIF-1α gene (HIF1A) has been found in several carcinomas, including ovarian cancer. Ovarian cancers are generally refractory to platinum-based chemotherapy. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells after chemotherapy are poorly defined. Genomic instabilities, such as copy number variation(CNV), may play an important role in chemoresistance and have been implicated in many complex diseases, like cancer.. We analyzed CNV data that is publically available through the Cancer Genome Atlas and others. Of particular interest was the transcription factor HIF1A which plays an integral role in oxidative stress response such as those induced by chemotherapy reagents. The present study provides evidence for the rare escape of tumor cells from drug-induced cell death by entering a non-cycling senescent state. We report the adaptive response of human ovarian surface epithelium cells to CoCl2, a chemical hypoxia-mimicking agent resulting in a senescent-like state of chemoresistant cells. The effect of the treatment was evaluated on CNV of HIF-1α gene expression, cell proliferation, survival, and tumor invasiveness. We show here that CNV duplication events of HIF1α results in an oxidative stress response in cells leading to chemoresistance through the induction of cellular senescence. Understanding the molecular events associated with chemoresistance will ultimately lead to better patient treatment and outcomes

Identifying Senescence as a Mode of Chemo Resistance in Ovarian Cancer

Current treatments of ovarian and breast cancer result in chemo resistance all too often. It has been hypothesized that senescence-a dormant condition associated with increased age and apoptosis- may play a role in the development of chemo resistance. We performed an in-vitro study with HOSE (carboplatin-sensitive), SKOV3 (chemo resistant ovarian cancer), and CAMA1 (chemo resistant breast cancer) cell lines, which were exposed to a variety of platinum-based treatments meant to model current cover clinically relevant scenarios in terms of tumor hypoxia. They were then stained for senescence in-vitro using B-gal, and analyzed for proliferation using the Cell Counting Kit 8, trypan blue dye exclusion, and survival plating, among other methods. Real-time quantitative PCR was used to determine relative levels of gene expression for classical apoptotic and senescent markers. Our results indicate that proliferation was temporarily halted in SKOV3 and CAMA1 after treatment. Cell proliferation later resumed in these cell lines while HOSE cell underwent apoptosis. Analysis of genetic tests (such as qPCR) also revealed that SKOV3 and CAMA1 had decreased gene expression of key genes that regulate apoptosis and senescence (such as p53 and CDK2). It can be concluded from this data that senescence was in fact a mode of chemoresistance and that future treatments may want to focus on disabling cancerous cells’ senescent stage

Strenuous Exercise Increases the Risk of Oxidative Stress in Ironman Triathlon Participants

Regular physical activity has been linked to greater overall health. Literature review and studies have also defined regular physical activity as a reducer of life-threatening illnesses such as cardiovascular disease, diabetes and obesity. However, long increments of strenuous exercise can produce oxidative stress and muscle fatigue in the human body. The increase in oxygen consumption during strenuous exercise leads to elevated reactive oxygen species (ROS). Cells continuously produce free radicals and reactive oxygen species as part of metabolic processes in the body. These free radicals are neutralized by an antioxidant defense system in the body consisting of enzymes, such as catalase, and non-enzymatic antioxidants. An Ironman Triathlon consists of a 2.4-mile (3.86 km) swim, a 112-mile (180.25 km) bicycle ride and a marathon 26.2-mile (42.2 km) run, raced in that order and without a break. It is widely considered by athletes to be one of the most demanding sporting events in the world. It is hypothesized that a physically challenging event such as the Ironman Triathlon can be linked to elevated cortisol levels, increased occurrence of DNA damage, elevated concentrations of ROS, and consequently increased oxidative stress in humans. In order to derive conclusive results regarding the hypothesis, groups containing athletes who completed the full Ironman race, the half Ironman race, and a control group of moderately active individuals were established and individuals were required to report Garmin Smartwatch health and wellness data. The half Ironman consists of a 1.2-mile (1.93 km) swim, a 56-mile (90.12 km) bicycle ride and a marathon 13.1-mile (21.1 km) run, raced in that order and without a break. Several protocols were then applied to derive data necessary to complete the research. After the participants were selected, their saliva was collected in a non-invasive fashion and was used in the Elisa Saliva Kit to determine cortisol concentration. The saliva samples were also utilized to perform DNA and RNA extraction; and the resulting products were analyzed for quantity and quality of the DNA and RNA. Real time PCR allows scientists to monitor PCR while it is occuring. In this technique, luminescence is produced by reporter molecules as the PCR products increase with every cycle. To determine ROS concentration, the ROS-Glo assay, which provides a light signal that is proportional to the ROS in a given sample, was utilized. An additional marker of oxidative stress is 8-oxo-2-deoxyguanosine(8-oxo-dG). The OxiSelect™ Oxidative DNA Damage ELISA uses antibody and antigen interactions to report the concentration of 8-oxo-dG in a sample. Furthermore, the results indicate an increase in enzymatic indicators of elevated ROS, elevated cortisol levels, and disruption of sleep in the participating athletes after the race. In conclusion, the athletes who completed the full Ironman triathlon experienced increased amounts of oxidative stress than their less active counterparts in the control group, as was denoted by the elevated cortisol levels, increased 8-oxo-dG concentrations, and increased ROS concentrations.Such a rigorous event negatively impacted participants and caused oxidative stress

Abstract 70: A role for the chromatin remodeling protein CHD3 in ovarian cancer therapy response

Carboplatin and cisplatin are chemotherapeutic agents that are used extensively for treating epithelial ovarian cancer. These drugs can be highly effective, yet tumors are frequently refractory to treatment or become resistant upon tumor relapse. Epigenetic silencing, particularly at promoter regions of genes regulates important cell function and has been associated with all stages of tumor formation and progression and may contribute to therapy response. We analyzed the epigenome of 50 primary ovarian tumors and 12 normal ovarian samples using an array based method previously developed in our lab and associated Affymetrix U133 expression data. We then identified gene candidates that segregate patients based on platinum sensitivity and patient survival. These candidates were then pooled into a genome-wide RNAi-based screen where we validated a gene encoding a chromatin remodeling protein, CHD3, a member of the Mi-2 NuRD complex, and show that it is linked to chemoresistance. CHD3 is silenced through an epigenetic mechanism in both ovarian cancer cell lines and primary ovarian tumors. When ovarian cancer cell lines that are transcriptionally silenced for CHD3 are challenged with carboplatin they display a striking slow growth phenotype as well as increased resistance to the chemotherapy drugs carboplatin and cisplatin. Taken together, we provide the first evidence for a role for CHD3 as an important mediator of chemoresistance in ovarian cancer. Furthermore, CHD3 might represent a response predictor and potential therapeutic target for predicting chemoresistance in this disease

Abstract 3144: A role for DOK2 methylation in platinum resistance and tumor suppression in ovarian cancer

Ovarian cancer is the 5th leading cause of cancer in women, affecting close to 22,000 women in the year 2011, of which nearly 15,500 will die. It is difficult to detect until it reaches advanced stages and becomes malignant. Currently, the standard treatment for ovarian cancer is platinum-based therapeutics, such as Carboplatin or Cisplatin, combined with Taxol. Unfortunately, approximately 25% of patients are inherently platinum-resistant and all patients who suffer from recurrence will have developed acquired platinum resistance. The genetic/epigenetic causes of this resistance are poorly understood. Epigenetic events are reversible and the identification of genes altered by this mechanism may lead to studies on how to reprogram the process leading up to resistance. To examine the ovarian epigenome, we utilized an array based method, Methylation Oligonucleotide Microarray Analysis (MOMA), to analyze a set of 50 primary ovarian tumors and 12 ovarian normal samples. We identified epigenetic differences that segregated with platinum response and then associated this with expression data to identify gene candidates transcriptionally repressed and methylated in patients resistant to platinum. Next, a pooled shRNA screen was performed on candidate genes to identify those that were functionally relevant to platinum resistance. One of the validated candidate genes identified through the pooled shRNA screen was DOK2, an adapter protein downstream of tyrosine kinase, which has been shown by others to be a lung cancer tumor suppressor. We show that suppression of DOK2 by short hairpin RNAs in ovarian cell lines conferred resistance to platinum treatment. To elucidate the mechanism for resistance, we measured the influx of platinum into the cells using C-14 tagged carboplatin. As a result, uptake of carboplatin was found to be decreased with DOK2 suppression. Consistent with DOK2 having tumor suppressor activity, knockdowns in ovarian cell lines increases growth and migration. Furthermore, loss of DOK2 induces invasive and tumorigenic phenotypes in ovarian cell lines. DOK2 is already a proven tumor suppressor in lung cancer, and our experiments indicate DOK2 has tumor suppressor features in ovarian cancer as well. We show that DOK2 is a tumor suppressor in ovarian cancer and that the loss of DOK2 also contributes to platinum resistance. Understanding DOK2 function will help us understand ovarian cancer development, progression as well as therapy resistance