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Our objective was to evaluate the impact of body composition (BC), beyond body mass index (BMI), and lipid metabolism disorders on therapeutic responses and outcomes in high-grade serous ovarian cancer (HGSOC).Methods: 123 and 415 patients with advanced HGSOC from two cohorts (PUC and TCGA) were analyzed. Databases containing clinical/genomic variables were built-up. BC was estimated using the measurement of adiposity (e.g., Whole body Adipose Tissue [WBAT]) and muscle mass (Lumbar muscle area to vertebral body at L4-level, [PLVI]) by CT-scan. A list of 425 genes linked to obesity and lipid metabolism was used to identify clusters using non-negative matrix factorization. GSEA, Gene Ontology, KEGG pathways enrichment, and Ecotyper analyzes were also made. Survival curves and Cox-regression models were also built-up. Results: We identified four BC types and two clusters that, unlike BMI, effectively correlate with survival regardless of achieving optimal debulking or complete response. Mention deserves central sarcopenia, which was associated with worse survival in any condition. So also, that recovering a normal BC and adding medications to correct metabolism disorders (e.g., statins) could have a positive impact on outcomes. Along with this, we showed that micro-environments depleted of immune cells predominate in HGSOC, something more evident in the BC type (High WBAT/Low PLVI) and cluster (Obesity/Lipid Metabolism Type I) with worse prognosis. Conclusions: Here, we demonstrate the relevance of BC and lipid metabolism disorders in determining therapeutic responses and long-term outcomes. Also, the importance of incorporating corrective measures addressing these disorders to obtain better results. (Research supported by Fondecyt 1201083) Citation Format: Mauricio A. Cuello-Fredes, Fernan Gomez, Ignacio Wichmann, Felipe Suarez, Sumie Kato, Jorge Brañes, Elisa Orlandini, Carolina Ibañez. Body composition and metabolic dysfunction really matter for the achievement of better outcomes in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 922.

Although obesity-associated metabolic disorders have a negative impact on various cancers, such evidence remains controversial for ovarian cancer. Here, we aimed to evaluate the impact of body composition (BC) and metabolism disorders on outcomes in high-grade serous ovarian cancer (HGSOC). Methods: We analyzed clinical/genomic data from two cohorts (PUC n = 123/TCGA-OV n = 415). BC was estimated using the measurement of adiposity/muscle mass by a CT scan. A list of 425 genes linked to obesity/lipid metabolism was used to cluster patients using non-negative matrix factorization. Differential expression, gene set enrichment analyses, and Ecotyper were performed. Survival curves and Cox-regression models were also built-up. Results: We identified four BC types and two clusters that, unlike BMI, effectively correlate with survival. High adiposity and sarcopenia were associated with worse outcomes. We also found that recovery of a normal BC and drug interventions to correct metabolism disorders had a positive impact on outcomes. Additionally, we showed that immune-cell-depleted microenvironments predominate in HGSOC, which was more evident among the BC types and the obesity/lipid metabolism cluster with worse prognosis. Conclusions: We have demonstrated the relevance of BC and metabolism disorders as determinants of outcomes in HGSOC. We have shone a spotlight on the relevance of incorporating corrective measures addressing these disorders to obtain better results.

Despite growing evidence linking obesity to the development and overall survival of cancer patients, the impact of increased body mass index (BMI) on gene expression patterns in gynecologic cancers is unclear. To address this, we herein downloaded RNAseq gene expression data from The Cancer Genome Atlas (TCGA) UCEC (uterine corpus endometrial carcinoma) dataset and matched it with clinical data entries including patient height and weight. We calculated BMI for each patient and used it to stratify patients as normoweight (BMI < 25), overweight (25 ≤ BMI < 30) or obese (BMI ≥ 30). BMI and nutritional status were matched to RNAseq gene expression data. Considering the known differences between endometrioid and serous uterine corpus tumors, analyses were performed separately for endometrioid (n = 356) and serous carcinomas (n = 117). Gene set enrichment analysis (GSEA) was performed between normoweight, overweight and obese patients. GSEA shows that tumors from obese patients display multiple dysregulated Gene Ontology (GO) terms (Biological Process, BP), which also differ between serous and endometrioid tumors. We further explored the impact of obesity on transcriptomic profiles of serous and endometrioid carcinomas by nutritional status independently by differential expression analysis using limma-voom. Significantly up- or downregulated genes were defined as those with logFC ≥ 1.5 at a non-adjusted P-value ≤ 0.01. Despite a clear enrichment or suppression of GO terms as evidenced by GSEA, transcriptomic analyses did not clearly separate patients according to their nutritional status. Therefore, obesity may impact on the dysregulation of biological processes through the cumulative effects of subtle differences in gene expression that, individually, do not seem to play a major role in oncogenesis. These results spotlight the relevance of obesity on the biology of uterine corpus cancers, which impact on the clinical course and treatment response of obese patients is yet to be determined. Funding: This work was supported by CONICYT FONDAP 15130011 (MC, IW), CONICYT-PFCHA/Doctorado Nacional 2019-Folio 21190421 (FG) and FONDECYT 1201083 (MC). Citation Format: Ignacio A. Wichmann, Fernán Gómez, Cristián Salazar, Felipe Suárez, Sumie Kato, Mauricio A. Cuello-Fredes. Distinct biological response of uterine corpus serous and endometrioid carcinomas to obesity may contribute to differences in clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3361.

To investigate whether specific obesity/metabolism‐related gene expression patterns affect the survival of patients with ovarian cancer. Clinical and genomic data of 590 samples from the high‐grade ovarian serous carcinoma (HGOSC) study of The Cancer Genome Atlas (TCGA) and 91 samples from the Australian Ovarian Cancer Study were downloaded from the International Cancer Genome Consortium (ICGC) portal. Clustering of mRNA microarray and reverse‐phase protein array (RPPA) data was performed with 83 consensus driver genes and 144 obesity and lipid metabolism‐related genes. Association between different clusters and survival was analyzed with the Kaplan–Meier method and a Cox regression. Mutually exclusive, co‐occurrence and network analyses were also carried out. Using RNA and RPPA data, it was possible to identify two subsets of HGOSCs with similar clinical characteristics and cancer driver mutation profiles (e.g. TP53), but with different outcome. These differences depend more on up‐regulation of specific obesity and lipid metabolism‐related genes than on the number of gene mutations or copy number alterations. It was also found that CD36 and TGF‐ß are highly up‐regulated at the protein levels in the cluster with the poorer outcome. In contrast, BSCL2 is highly up‐regulated in the cluster with better progression‐free and overall survival. Different obesity/metabolism‐related gene expression patterns constitute a risk factor for prognosis independent of the therapy results in the Cox regression. Prognoses were conditioned by the differential expression of obesity and lipid metabolism‐related genes in HGOSCs with similar cancer driver mutation profiles, independent of the initial therapeutic response.

// Rafaela Erices 1, 2 , Sofia Cubillos 2 , Raul Aravena 2, 3 , Felice Santoro 2 , Monica Marquez 2 , Renan Orellana 1, 15 , Carolina Ramirez 2 , Pamela Gonzalez 2, 4 , Patricia Fuenzalida 2 , Maria Loreto Bravo 2, 4, 5 , Barbara Oliva 2, 4 , Sumie Kato 1 , Carolina Ibanez 5, 6, 7 , Jorge Branes 1 , Erasmo Bravo 8 , Catalina Alonso 8 , Karen Garcia 7, 9 , Clemente Arab 10 , Vicente A. Torres 11, 16 , Alejandro S. Godoy 2, 12 , Jaime Pereira 6 , Galdo Bustos 13 , Julio Cesar Cardenas 13, 14 , Mauricio A. Cuello 1 , Gareth I. Owen 2, 4, 5, 7, 16 1 Division of Obstetrics and Gynecology, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 2 Department of Physiological Sciences, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile, Santiago, Chile 3 Universidad Santo Tomas, Santiago, Chile 4 Biomedical Research Consortium of Chile, Santiago, Chile 5 Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Catolica de Chile, Santiago, Chile 6 Hematology and Oncology Department, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 7 Center UC Investigation in Oncology, Pontificia Universidad Catolica de Chile, Santiago, Chile 8 Hospital Gustavo Fricke, Vina de Mar, Santiago, Chile 9 Hospital Sotero del Rio, Santiago, Chile 10 Hospital Luis Tisne, Santiago, Chile 11 Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile 12 Department of Urology, Roswell Park Cancer Institute, Buffalo, NY, USA 13 Anatomy and Developmental Biology, Institute of Biomedical Science, Geroscience Center for Brain Health and Metabolism, University of Chile, Santiago, Chile 14 Buck Institute for Research on Aging, Novato, CA, USA 15 Universidad Bernardo OHiggins, Facultad de Salud, Departamento de Ciencias Quimicas y Biologicas, General Gana, Santiago, Chile 16 Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago, Chile Correspondence to: Gareth I. Owen, email: gowen@bio.puc.cl Keywords: thrombocytosis, hemostasis, EA.hy926, SKOV3, UCI101 Received: July 27, 2016 Accepted: January 27, 2017 Published: February 15, 2017 ABSTRACT Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.

Objectives HGSOC is the deadliest gynecologic cancer. Molecular analysis to samples available at The Cancer Genome Atlas (TCGA) allowed the identification of 4 molecular subtypes with different biology/prognosis. Recently, our group demonstrated by bioinformatic analysis that patients expressing high levels of genes related to obesity and lipid metabolism (e.g. CD36/TGFs) experienced poorer outcomes in two international cohorts (TCGA and AOCS). Here, our goal was to establish if there was correlation between such patterns and the prevalence and prognosis of different molecular subtypes of HGSOC. Methods We retrieved matching data of 455 cases of stage II-IV HGSOC from TCGA and categorized into 4 clusters based first on molecular subtypes (differentiated, immunoreactive, mesenchymal and proliferative) and then subdivided based on obesity and lipid metabolism gene pattern expression (CD36/TGFs high vs low). Proportion and survival analyses were carried out. Chi square, Kaplan Meier were used to assess statistical significance. Results Mesenchymal subtype was significantly more prevalent among women expressing CD36/TGFs high (51,2% vs 6,9%, p Conclusions Our results confirm the importance of immune response and the negative impact that obesity and lipid metabolism alterations have in defining prognosis in HGSOC (supported by Fondecyt 1160800).

Background High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histologic type of ovarian cancer. To date, there are no reliable biomarkers to effectively predict patient prognosis. Studies have demonstrated inflammation and tumor infiltrating lymphocytes (TILs) correlate with a bad and good prognosis, respectively. Here, we sought to evaluate systemic inflammation and TILs as early prognostic markers of survival. Methods Neutrophil-to-lymphocyte ratio (NLR) and serum Lactate Dehydrogenase (LDH) were used as indicators of systemic inflammation. NLR, serum LDH, tumor infiltrating lymphocytes (TILs), PDL1 and quality of debulking surgery were evaluated as determinants of progression-free survival (PFS) and overall survival (OS) in a cohort of 128 HGSOC patients. Results Initial univariate analysis showed that systemic inflammation measures (NLR and serum LDH), debulking surgery, and intra-epithelial TILs have a significant impact on both PFS and OS. After adjustment for several variables, multivariate analyses confirmed intraepithelial CD4+ T-cells, systemic inflammation measures, PDL1 and debulking surgery as determinants of better OS and PFS. Conclusions Systemic inflammation and TILs are early determinants of OS in HGSOC. Other variables such as the quality of debulking surgery and PDL1 also improve survival of patients. Regarding TIL sub-populations, intraepithelial CD4+ cells are associated to an increase in both PFS and OS. We also confirmed previous reports that demonstrate intraepithelial CD8+ cells correlate with an increase on PFS in ovarian cancer. A combined score using systemic inflammation and TILs may be of prognostic value for HGSOC patients.

Cell plasticity of ‘stem-like’ cancer-initiating cells (CICs) is a hallmark of cancer, allowing metastasis and cancer progression. Here, we studied whether simvastatin, a lipophilic statin, could impair the metastatic potential of CICs in high-grade serous ovarian cancer (HGS-ovC), the most lethal among the gynecologic malignancies. qPCR, immunoblotting and immunohistochemistry were used to assess simvastatin effects on proteins involved in stemness and epithelial-mesenchymal cell plasticity (EMT). Its effects on tumor growth and metastasis were evaluated using different models (e.g., spheroid formation and migration assays, matrigel invasion assays, 3D-mesomimetic models and cancer xenografts). We explored also the clinical benefit of statins by comparing survival outcomes among statin users vs non-users. Herein, we demonstrated that simvastatin modifies the stemness and EMT marker expression patterns (both in mRNA and protein levels) and severely impairs the spheroid assembly of CICs. Consequently, CICs become less metastatic in 3D-mesomimetic models and show fewer ascites/tumor burden in HGS-ovC xenografts. The principal mechanism behind statin-mediated effects involves the inactivation of the Hippo/YAP/RhoA pathway in a mevalonate synthesis-dependent manner. From a clinical perspective, statin users seem to experience better survival and quality of life when compared with non-users. Considering the high cost and the low response rates obtained with many of the current therapies, the use of orally or intraperitoneally administered simvastatin offers a cost/effective and safe alternative to treat and potentially prevent recurrent HGS-ovCs.

Our laboratory has implemented an in vitro assay to estimate the response to chemotherapy in ovarian cancer cells pertaining to individual patients. In two selected patients, we determined the correlation between an in vitro assay of cells from suspected ovarian cancer ascites, with the clinical chemotherapy response. Cancer cells isolated from peritoneal fluid with suspected ovarian cancer were tested for cytotoxicity with corresponding chemotherapy regimens. Circulating Cal25 levels and attending physician consultation determined clinical course and response to chemotherapy. The in vitro assay result correlated with Cal25 levels, progression free survival and attending physician evaluation. The assay predicted correctly the failure of two successive chemotherapy regimes in the first patient, while predicting a favorable clinical response in the second subject.

// Sumie Kato 1 , Lorena Abarzua-Catalan 1 , Cesar Trigo 1 , Ana Delpiano 1 , Cristobal Sanhueza 2 , Karen Garcia 1 , Carolina Ibanez 2 , Katherine Hormazabal 1 , Daniela Diaz 1 , Jorge Branes 1 , Enrique Castellon 4 , Erasmo Bravo 5 , Gareth Owen 3 and Mauricio A. Cuello 1 1 Division of Obstetrics and Gynecology, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 2 Department Hematology and Oncology, Pontificia Universidad Catolica de Chile, Santiago, Chile 3 Department of Physiological Sciences, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile, Santiago, Chile 4 Faculty of Medicine, University of Chile, Santiago, Chile 5 Gynecologic Oncology Unit, Hospital Gustavo Fricke, Vina del Mar, Chile Correspondence to: Mauricio A. Cuello, email: // Keywords : leptin, ovary, neoplasia, metastasis, obesity Received : March 10, 2015 Accepted : May 13, 2015 Published : May 22, 2015 Abstract The evidence linking obesity with ovarian cancer remains controversial. Leptin is expressed at higher levels in obese women and stimulates cell migration in other epithelial cancers. Here, we explored the clinical impact of overweight/obesity on patient prognosis and leptin’s effects on the metastatic potential of ovarian cancer cells. We assessed clinical outcomes in 70 ovarian cancer patients (33 healthy weight and 37 overweight) that were validated with an external cohort from The Cancer Genome Atlas (TCGA) database. Progression-free and overall survival rates were significantly decreased in overweight patients. Similarly, a worse overall survival rate was found in TCGA patients expressing higher leptin/OB-Rb levels. We explored serum and ascites leptin levels and OB-Rb expression in our cohort. Serum and ascites leptin levels were higher in overweight patients experiencing worse survival. OB-Rb was more highly expressed in ascites and metastases than in primary tumors. Leptin exposure increased cancer cell migration/invasion through leptin-mediated activation of JAK/STAT3, PI3/AKT and RhoA/ROCK and promoted new lamellipodial, stress-fiber and focal adhesion formation. Leptin also contributed to the maintenance of stemness and the mesenchymal phenotype in ovarian cancer cells. Our findings demonstrate that leptin stimulated ovarian cancer cell migration and invasion, offering a potential explanation for the poor prognosis among obese women.

Progesterone in hormone replacement therapy (HRT) preparations increases, while hysterectomy greatly reduces, the incidence of breast cancer. Cross-talk between the progesterone and growth factor signaling pathways occurs at multiple levels and this maybe a key factor in breast cancer survival and progression. To test this hypothesis, we characterized the effect of progesterone pre-treatment on the sensitization of the epidermal growth factor (EGF) signaling pathway to EGF in the breast cancer cell line ZR-75. For the first time in ZR-75 cells and in agreement with previous work using synthetic progestins, we demonstrate that pre-treatment with the natural ligand progesterone increases EGF receptor (EGFR) levels and subsequent ligand-dependent phosphorylation. Downstream we demonstrate that progesterone alone increases erk-1 + 2 phosphorylation, potentiates EGF-phosphorylated erk-1 + 2 and maintains these levels elevated for 24 h; over 20 h longer than in vehicle treated cells. Additionally, progesterone increased the levels of STAT5, another component of the EGF signaling cascade. Progesterone increased EGF mediated transcription of a c-fos promoter reporter and the nuclear localization of the native c-fos protein. Furthermore, progesterone and EGF both alone and in combination, significantly increase cell proliferation. Several results presented herein demonstrate the conformity between the action of the natural ligand progesterone with that of synthetic progestins such as MPA and R5020 and allows the postulation that the progestin/progesterone-dependent increase of EGF signaling provides a survival advantage to burgeoning cancer cells and may contribute to the breast cancer risk associated with endogenous progesterone and with progestin-containing HRT.

Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process. Materials & Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT). Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry. Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers. Funding: FONDECYT 1120292, 3150028 & 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001 Citation Format: Andres Valdivia, Dusan Racordon, Raul Aravena, Gabriel Mingo, Alejandra Sandoval, Maria Loreto Bravo, Mauricio A. Cuello, Sumie Kato, Rafaela Erices, Carolina Ramirez, Pamela Gonzalez, Beatriz Sanchez, Alejandro H. Corvalan, Gareth I. Owen. Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2017-789

Ovarian cancer metastasis occurs when malignant cells migrate from the primary tumor into the ascitic fluid and form a secondary tumor in the peritoneal cavity. The cells responsible for this process, Metastases Initiating Cells (MICs), suffer a series of changes in protein expression, with increased Epithelial Mesenquimal Transition and stem cell markers having been reported. Tissue Factor Pathway Inibitor-2 (TFPI-2) is a serine proteinase inhibitor and potential tumor suppressor gene, whose reduction is correlated with more metastases and poor prognosis. As the exact stage of TFPI-2 loss is still unknown, the purpose of our study was to determine TFPI-2 during ovarian cancer progression. Protein and RNA levels were assessed in primary tumor and metastatic tissue from papillary serous carcinoma by immunohistochemistry and qPCR, respectively. TFPI-2 RNA and protein levels were analyzed in cancer cell lines and primary cultured cancer cells isolated from ascitic fluid of advanced ovarian cancer patients and in cancer spheres derived from these cultures. Epigenetic changes of 3 CpG were analized by bisulfite and pirosequencing technique in ovarian cancer cell lines and in cancer spheres derived from these cultures. We report that significantly decreased TFPI-2 protein levels in metastatic tissue compared to the primary tumor, a result confirmed in matched primary and metastatic lesions from the same ovarian cancer patients. Furthermore, cancer spheres (representing potential MICs) displayed significantly lower levels of TFPI-2 than corresponding cell line and some primary cultured cancer cells. The cancer spheres showed a modest increase in methylation percentage in three specific CpG sites within the TFPI-2 promoter. Our results suggest that TFPI-2 loss plays a role in the cancer cell escape from the primary tumor. Future studies will determine the mechanism and the biological consequences of the progressive loss of TFPI-2 in cancer. Funding: FONDECYT 11140657, 1160800, 1140970, 3140335, 3150028, CORFO L2 13IDL2-18608, IMII-P09/016-F, CONICYT-FONDAP 15130011, BMRC 13CTI21526-P6 Citation Format: Jacqueline Fry, Sumie Kato, Lorena Abarzua, Pamela Gonzalez, Carolina Ramirez, Elisa Cumsille, Erasmo Bravo, Raimundo Correa, Juan E. Leiva, Cesar Paredes, Juan C. Roa, Carolina Ibañez, Mauricio Cuello, Gareth I. Owen, Maria L. Bravo. Regulation of TFPI-2 in the progression of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2017-5516

Introduction: High-grade serous ovarian cancer remains as the most lethal gynecologic cancer. Despite achieving optimal debunking using ultra-radical surgery followed by platinum-based chemotherapy, in the long term, most of the patient will die from their disease, mainly due to extensive carcinomatosis, bowel obstruction and cachexia. Recently our group has provided biological evidence supporting the role of obesity in impairing ovarian cancer prognosis. Obesity is considered as a chronic low-grade inflammation state where leptin can play a major role in modulating cancer behavior. In the present work we explore the effects of leptin levels found in ascites, comparing lean and obese population, in cytokine profile released from macrophages and cancer cells and its impact in macrophage behavior and invasiveness of ovarian cancer cells. Methods: Ascites from patients undergoing primary cytoreductive surgery for advanced stage high-grade serous ovarian cancer were collected to establish primary tissue cultures and measuring leptin levels by ELISA kit. A database was built up for clinical variables, and patients were stratified by body mass index (BMI) using the WHO classification. Three ovarian cancer cells (e.g. HEY, UCI 101 and SKOV3) were used to establish in vitro spheroids and assessing the effects of leptin alone or conditioned media (CM) collected from leptin-treated macrophages in migration/invasion of cancer cells in matrigel Boyden chamber assays. IL-6, IL-10 and IL-12 release were measured in both cancer cells and macrophages upon leptin treatment, at similar concentrations than those found in ascites collected from lean, overweight and obese patients. Monocyte THP-1 cells were PMA-transformed into macrophages and the IL-10/IL-12 release ratio was used to identify M1 or M2 polarization induced either by leptin alone or CM collected from leptin-treated cancer cells. Results: Ascites collected from obese patients express significantly higher leptin levels compared with ascites from lean patients (13 ±4 ng/ml vs. 4±2 ng/ml; p Conclusions: Our findings support a role of leptin as one of the factors driving the detrimental effect of obesity in high-grade serous ovarian cancer. Leptin induces a pro-inflammatory reinforcement loop between cancer cell and macrophage that favors ovarian cancer progression by turning the macrophage tumor-suppressive phenotype into a pro-tumorigenic one and prompting an inflammatory microenvironment. (Research funded by Fondecyt 1120292). Citation Format: Mauricio A. Cuello, Lorena Abarzua-Catalan, Sumie Kato, Isidora Solar, Claudia Arancibia. Leptin induces a pro-inflammatory macrophage-cancer cell reinforcement loop that favors high-grade serous ovarian cancer progression among overweight/obese women. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B61.

2-Methoxyestradiol (2ME) is an endogenous metabolite of 17β-estradiol with antiangiogenic and antitumor properties, although its mechanisms of action remain unclear. Progestins in hormone replacement therapy increase the risk of breast cancer. Progesterone also enhances the procoagulant activity and invasive potential of progesterone receptor (PR)-positive breast cancer cell lines, an effect largely mediated by induction of tissue factor (TF), the cellular activator of the coagulation cascade. Here we show that 2ME abrogates the induction TF expression in progesterone-treated breast cancer cells via a mechanism that does not involve the estrogen receptor. Instead, we demonstrate that by selectively antagonizing ERK1/2 signaling in breast cancer cells, 2ME limits the transactivation potential of ligand-bound PR and inhibits the expression of endogenous progesterone targets, such as TF and signal transducer and activator of transcription 5. We further demonstrate that 2ME can alter the phosphorylation status of PR. Thus, 2ME prevents progesterone-dependent increase in breast cancer cell invasiveness and procoagulant activity by uncoupling PR from the ERK1/2 signal transduction pathway.

Cancers of the reproductive tract account for 12% of all malignancies in women. As previous studies have shown that oestrogen metabolites can cause apoptosis, we characterised the effect of oestrogen and oestrogen metabolites on non-cancerous and cancerous human endometrial cells. Herein, we demonstrate that 2-methoxyoestradiol (2ME), but not 17β-oestradiol, induces apoptosis in cancer cell lines and primary cultured tumours of endometrial origin. In contrast, 2ME had no effect on cell viability of corresponding normal tissue. This ability of 2ME to induce apoptosis does not require oestrogen receptor activation, but is associated with increased entry into the G2/M phases of the cell cycle and the activation of both the intrinsic and the extrinsic apoptotic pathways. The selective behaviour of 2ME on cancerous as opposed to normal tissue may be due to a reduction in 17β-hydroxysteroid dehydrogenase type II levels in cancer cells and to a differential down-regulation of superoxide dismutase. Furthermore, we demonstrate that pre-treatment with 2ME enhances the sensitivity of reproductive tract cancer cells to the apoptotic drug tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), without the loss in cell viability to normal cells incurred by currently chemotherapeutic drugs. In conclusion, 2ME, alone or in combination with TRAIL, may be an effective treatment for cancers of uterine origin with minimal toxicity to corresponding healthy female reproductive tissue.

Introduction: The current knowledge in ovarian epithelial carcinogenesis grants a major role for chronic inflammation in driving the multiple steps in the natural history of the most deadly gynecological cancer. Different environmental stimuli have been associated with an increased ovarian cancer risk. Growing evidence has become to support the potential role of obesity as one of the conditioning factors. Leptin, an adipokine, controlling metabolism and energy expenditure and normally circulating at higher serum levels among overweight women, has been syndicated as the pro-inflammatory mediator in obesity. So far, few are known about the role of and how leptin modulate cells to cells interactions among the different components of tumor microenvironment. Goal: In the present study we investigated the leptin effect in prompting a pro-inflammatory microenvironment, the communication between macrophages and ovarian cancer cells and the impact of this interaction in cancer cell migration and invasion. Methods: HEY ovarian cancer cell line and the THP-1 monocyte cell line were used. THP-1 cells were differentiated to macrophages by using PMA (Phorbol 12-myristate 13-acetate) 200 nM for 24h. Upon differentiation macrophages were exposed to a time course of leptin (100 ng/ml, 6h to 24h). Upon control or leptin exposure, conditioned medium was collected from macrophages (MCM), and used to measure IL-6 release to the medium and to treat HEY cell line for 24h. Secreted IL-6 was used as inflammation marker and measured by ELISA assay. Protein extracts were obtained from HEY cells upon control and MCM exposure and used to quantify epithelial mesenchymal transition (EMT) marker vimentin by Immunoblotting and Immunocytochemistry. The effects of leptin and MCM in HEY migration and invasion was assessed by Boyden chamber assay without or with matrigel, respectively. Results: Upon leptin treatment both HEY and macrophages significantly increased IL-6 release to the medium. More importantly, incubation of MCM with HEY cells resulted in a synergistic increase of IL-6 release, (from a basal release of 13,4±2 pg/mL to 2,3×104 pg/mL upon MCM exposure). Moreover, HEY cells exposure to MCM resulted in phenotypic changes that were associated to changes in vimentin expression (mainly upon exposure to 6h leptin incubated MCM). Finally, this exposure to leptin-incubated MCM significantly increased migration and invasion of HEY cells. Conclusions: Our findings support a role of leptin in prompting a pro-inflammatory microenvironment that involved an increase in IL-6 release from the macrophages. These changes induced by leptin in macrophages resulted in a cytokine secretion pattern, which consequentially modified cancer cells phenotype favoring migration and invasion and a more aggressive epithelial ovarian cancer behavior. (Funding: FONDECYT 1120292 and 3140335) Citation Format: Sumie Kato, Lorena Abarzua-Catalan, Isidora Solar-Costabal, Karen García, Jorge Branes, Gareth I. Owen, Mauricio A. Cuello. Leptin induces an IL-6 mediated interaction between macrophages and ovarian cancer cells that prompted invasiveness and migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2395. doi:10.1158/1538-7445.AM2015-2395

Ovarian cancer patients respond well to first line chemotherapy but present high incidences of toxicity followed by consistent relapse within 18 months. Despite every cancer and patient being unique, generally all cancers are treated by the same chemotherapy regimes and patient metabolism is not considered in treatment selection. Differing metabolism may account for unacceptable toxicity or failures in chemotherapy treatment. It is becoming accepted that the metastatic cell is distinct to those found in the primary tumor and is thus the true target of chemotherapy. Our objectives were to isolate these potential ovarian metastatic cells from patients and correlate their in vitro response to the chemotherapy with clinical response. The single nucleotide polymorphism (SNP) distribution of genes related to chemotherapy metabolism was also analyzed in each patient. Primary cultures and blood samples of ovarian patients were obtained with signed informed consent from a network of Chilean and Peruvian hospitals. Chemotherapy response was assessed by MTS and data correlated with medical follow-up. SNPs were detected using commercial taqman probes from Applied Biosystem. Results. Ovarian cancer patients with clinical follow-up demonstrated a marked separation of progression free survival (PFS) curves according to classification within our in vitro assay. Analysis of exclusively first line patients demonstrated a median PFS of 17 months for patients predicted to respond to treatment showed by our assay, but only an average of 2 months of PFS in patients that the assay predicted no response. Initial analysis of 14 SNPs related to paclitaxel-platinum based chemotherapy demonstrates marked differences in Chilean patients. Conclusion. We demonstrate a clear distinction in time to relapse between the patients predicted or not to respond to treatment by our assay. Ovarian cancer patients demonstrate notably different distributions in SNPs previously correlated with platinum metabolism. Our results highlight the requirement of the medical community to take this research to the stage of randomized clinical trials. CORFO 13CTI21526-P6 & 13IDL2-18608, FONDECYT 1140960, 1140657 & 1120292. Citation Format: Maria Loreto Bravo, Pamela Gonzalez, Sumie Kato, Carolina Ibañez, Marcelo Garrido, Jorge Brañes, Maria Isabel Barriga, Eva Bustamante, Nicanor Barrena, Catalina Alonso, Leonel Muñoz, Erasmo Bravo, Clemente Arab, Alejandro Barra, Paula Jimenez, Patricio Gayan, Fernando Gonzalez, Ignacio Chavez, Alfredo Aguilar, Joseph Pinto, Mauricio Cuello, Gareth Owen, Jaime Cartagena. Prediction of chemotherapy response and metabolism for the tailoring of ovarian cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2015-4314

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2. Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue.

Introduction: An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor correlated with bad prognosis and metastasis, however the exact role that platelets play in tumor progression has not been fully elucidated. The expression of Tissue Factor (TF) protein by cancer cells has been shown to correlate with Hypercoagulable state and metastasis. It is speculated that cancer cells need to acquire a “Metastasis Initiating Cell (MIC)” phenotype. To achieve this, cells may suffer an Epithelial to Mesenchymal Transition (EMT) coupled with stem-like protein expression in which E-cadherin is lowered, while CD44 and N-cadherin are increased. Ovarian cancer patients develop ascitis, a build up of peritoneal fluid that contains cancerous and serum derived cells. After the surgical removal of the primary and visible tumors, these ascitic borne cells form peritoneal tumors and thus make a good model to study platelet/cancer cells interaction in metastasis. The aim of this work was determine the effect of platelet-cancer cell interaction on TF and EMT marker levels and cancer cell migration in ascites-derived cancer cells. Material and Methods: Ovarian ascitic-isolated cancer cells and cell lines were co-cultivated with human washed platelets (from healthy donors). TF and EMT marker levels were determined by Western blotting. Cancer cell migration was determined by Boyden chambers and the scratch assay. Results: The co-culture of ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) increases TF protein levels and activity, 3) induces EMT markers, 3) promotes chemoattraction and cancer cell migration. Conclusion: We present evidence that platelet-cancer cell interaction promotes a Metastasis Initiating Cell phenotype and enhanced migration. We speculate that platelet-cancer cell interaction may promote the formation of metastatic foci worsening the patient prognosis. Fundings: FONDECYT 1120292, 3120003, 1100870, CORFO 13IDL2-18608, BMRC 13CTI-21526-P6 Citation Format: Renan F. Orellana, Barbara Oliva, Sumie Kato, Loreto Bravo, Pamela Gonzalez, Olga Panes, Diego Mezzano, Mauricio Cuello, Gareth Owen. Platelet interaction induces Tissue Factor and a pro-metastatic phenotype in ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1117. doi:10.1158/1538-7445.AM2014-1117

Ovarian cancer is the most deadly gynecological malignancy. Its lethality is mainly due to late diagnosis and acquisition of resistance to available therapies. Current evidence considers “tumor cell heterogeneity” as a cancer hallmark. A subset of cancer cells, named "cancer-initiating cells" (CICs), possesses stem cell-like capabilities allowing self-renewal, differentiation and dedifferentiation into other cell types, some of them evolved to a more invasive and resistant phenotype. Thus, the existence of CICs allows tumor to overcome therapy and regrowth as resistant kind over time. Finding therapeutic strategies effective against this subset of cells remain a major goal in cancer therapeutics. Previously we have shown that 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, induces cell death in ovarian cancer cells. Objective: In the present work we investigated the potential effects of 2-ME on CICs isolated from either ovarian cancer cell lines or primary tissue cultures obtained from ovarian cancer patients. Methods: CICs were isolated from both ovarian cancer cell lines (SKOV3, UCI 101, HEY) as well as primary tissue cultures established from ascites samples. Stemness, epithelial-mesenchymal transition (EMT), and anti-apoptotic markers were studied by immunoblotting (W-B), RT-PCR and immunohistochemistry in CICs cultured under stem-selection medium/non-attachment conditions and after treatment with different concentrations of 2-ME (5 to 10 uM, up to 24h). CICs treated under similar conditions were then exposed to different concentrations of chemotherapeutic reagents (i.e. paclitaxel, 0,5-5 uM, for up to 48 h). Cell death was measured by Tunel assay and PARP cleavage (measured by W-B). Results: Isolated CICs grow forming ovospheres that express higher levels of Oct-4, Nanog, Snail, N-Cadherin, CD44, CD133, survivin, and lower levels of E-cadherin compared with non-selected cell population (whole cell population, WP). In contrast to WP, CICs did not undergo apoptosis upon 2-ME exposure (5 uM for 24 h) measured by PARP cleavage. However, 2-ME treatment results in a decrease of Nanog, N-cadherin, Snail, Survivin and an increase in E-cadherin protein expression levels. In addition, a decrease in mRNA levels of Twist, Zeb2 (but not Zeb1) is observed under similar conditions (5 uM for 6 h). 2-ME pretreatment increases paclitaxel-induced cell death in CICs compared to those treated with paclitaxel alone, measured by Tunel assay. Conclusions: 2-ME induces the loss of stemness and EMT markers in epithelial ovarian CICs. This differentiating effect induced by 2-ME results in sensitization of this subset of resistant cells to chemotherapeutic agents. Further studies might be conducted to assess the clinical usefulness of this combination. (FONDECYT 1120292, and BMRC CTU06) Citation Format: Mauricio A. Cuello, Sumie Kato, Katherine Hormazábal, Lorena Abarzua, Loreto Bravo, Isabel Barriga, Nicanor Barrena, Jorge Brañes, Erasmo Bravo, Roger Gejman, Enrique Castellón, Gareth Owen. 2-Methoxyestradiol induces differentiation of epithelial ovarian cancer-initiating cells sensitizing them to chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3730. doi:10.1158/1538-7445.AM2013-3730

Introduction: The treatment of ovarian cancer is consists principally in radical surgery followed by chemotherapy. Patients generally respond well to first line chemotherapy (70%) but consistently relapse within 18 months, with successive rounds of chemotherapy delivering extremely low patient benefit. Despite every cancer being unique, generally all cancer patients are treated by the same chemotherapy regimes. In previous decades an approach has been to use chemosensitivity assays based on cells isolated from solid tumors to predict chemotherapy response. However, it the metastatic cancer cell (not cells in the primary tumor) that is the true target of chemotherapy. In our laboratory we have resurrected the concept of chemosensitivity but focusing exclusively on cells that have escaped the primary tumor and are potentially enriched in stem-cell like properties. Our objective was to isolate potential ovarian metastatic cells from patients and coorelate their in vitro response to the chemotherapy observed in the patient. Materials and methods: Primary cultures of ovarian cancer ascites (peritoneal fluid) were obtained with signed informed consent from a network of Chilean hospitals and potential metastatic cells cultivated. Chemotherapy response was assessed by MTS and data correlated with CA125 levels, imaging and medical criteria. Results: Analysis of 39 ovarian cancer patients with clinical follow-up demonstrated an average progression free survival of 18 months for patients predicted to respond by our assay, but only 2 months in predicted non-responders. The assay corrected identified 5 of the 6 patients who did not respond to first line carboplatin-paclitaxel therapy. Discussion: We demonstrate a clear distinction in time to relapse between patients predicted to respond in our assay as opposed to patients with no predicted clinical response, demonstrating the requirement to take this research to the stage of randomized clinical trials. Funding BMRC CTU06 & FONDECYT 3120003 and 1120292 Citation Format: Maria L. Bravo, Pamela Gonzalez, Sumie Kato, Marcelo Garrido, Jorge Brañes, Javier Pizarro, Maria Isabel Barriga, Hector Leon, Eva Bustamante, Catalina Alonso, Erasmo Bravo, Miguel Angel Celis, Barbara Aguilera, Alejandro Barra, Nicanor Barrena, Carolina Ibañez, Paula Jimenez, Mauricio Cuello, Gareth Owen. Personalized cancer therapy for ovarian cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1187. doi:10.1158/1538-7445.AM2013-1187

Background An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and “Metastasis Initiating Cell (MIC)” marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. Methods With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. Results The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. Conclusions We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1304-z) contains supplementary material, which is available to authorized users.