bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B49: The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer
Authors: Belotte Jimmy , Fletcher Nicole , Awonuga Awoniyi , Alexis Mitchell , Abu-soud Husam , Diamond Michael , Saed Mohammed , Saed Ghassan
Published: August 2014

Objective: To investigate the role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer (EOC).

Methods: Two parent EOC cell lines (MDAH-2774 and SKOV-3) and their chemoresistant counterparts were used. Total RNA was extracted and subjected to real-time RT-PCR to evaluate the expression of inducible nitric oxide synthase (iNOS) and glutathione reductase (GSR) in all cells. Nitrate/nitrite levels and GSR activity were also measured using colorimetric and ELISA assays respectively. Cell proliferation was assessed using Ki-67 immunostaining in all cells. Unpaired t-tests were used for statistical analysis at p-value <0.05 for significance.

Results: Both EOC cisplatin sensitive cells manifested significant increase in the expression and activity of GSR as compared to their resistant counterparts (p<0.05). On the other hand, EOC sensitive cells manifested a decrease in iNOS expression as compared to their resistant counterparts (p<0.05). Nitrate/nitrite levels were significantly decreased in EOC sensitive cells as compared to their resistant counterparts (p<0.05). Ki-67 immunofluorescence staining revealed increased proliferation in cisplatin resistant cells compared to controls for the MDAH-277a and SKOV-3 cell lines.

Conclusion: Our data suggests that the development of cisplatin resistance heighten the prooxidant state in EOC leading to a further increase in cell proliferation.

Citation Format: Jimmy Belotte, Nicole Fletcher, Awoniyi Awonuga, Mitchell Alexis, Husam Abu-soud, Michael Diamond, Mohammed Saed, Ghassan Saed. The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B49.
Abstract B51: Management of ascites via the alfa-pump closed system in platinum-resistant-ovarian-cancer (PROC): A model of sequential non-invasive tumor-cell sampling through the urinary bladder
Authors: Fotopoulou Christina , Spiers Laura , Adjogatse Delali , Pickford Emily , Dina Roberto , Blagden Sara , Habib Nagy , Gabra Hani
Published: August 2014

Background: Malignant ascites in PROC is a therapeutic dilemma. The Sequana-Medical alfapump-System (AP), a remotely controlled device connecting the patients'peritoneal cavity to their urinary bladder, has been evaluated for ascites in liver cirrhosis, but not as yet for malignant ascites.

Methods: We implanted the AP in the peritoneal cavity of two heavily pretreated PROC-patients (67y and 70y) who required repetitive ascites drainages monthly. We cross correlated the electronic download of recorded volume pumped (intraperitoneally→bladder) with weekly ultrasound, monthly cystoscopies and QoL-evaluation. Early morning urine for evaluation of cytology and tumor-cell molecular analysis was collected weekly.

Results: The implantation was under general anesthesia in a 60- and 35-minute procedure. The pump, draining 350ml – 500ml ascites/day successfully drained the ascites to dryness after 3 days (1stpatient) and 8 days (2ndpatient). The patients did not report pollakiuria or dysuria, merely an increased micturition volume. The 1st patient died of disease progression 2 months later; the 2nd patient we noticed a gradual amelioration of peripheral edemas and overall performance-status within 3 weeks after insertion and was able to commence weekly chemotherapy with paclitaxel. Histopathological analysis of the urine revealed rich malignant cell content; this was used to create FFPE-cell-blocks for molecular- pathological profiling with sequential Caris-Target-Now-analysis and full exome-sequencing. QoL as measured by the EORTC QlQ-C30 and EORTC QLQ-OV28 showed high interindividual differences and only minimal influence by the pump.

Conclusion: This innovative approach addresses an area of unmet need for the control of malignant ascites and provides a non-invasive method of collecting tumor tissue for continuous molecular tumor characterization. A EUTROC-multicenter randomized-trial (AMAZE) is planned for evaluation of clinical and translational implications in PROC.

Citation Format: Christina Fotopoulou, Laura Spiers, Delali Adjogatse, Emily Pickford, Roberto Dina, Sara Blagden, Nagy Habib, Hani Gabra. Management of ascites via the alfa-pump closed system in platinum-resistant-ovarian-cancer (PROC): A model of sequential non-invasive tumor-cell sampling through the urinary bladder. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B51.
Abstract B65: The mitochondrial antioxidant Superoxide Dismutase 2 is necessary for ovarian cancer spheroid formation
Authors: Hemachandra LP Madhubhani , Dier Usawadee , Uusitalo Larissa M. , Hempel Nadine
Published: August 2014

The formation of cellular spheroid aggregates in the Intraperitoneal Cavity (IP) is considered to be an important step in the metastatic progression of ovarian cancer. Spheroids may represent a unique morphological adaptation that enhances long-term survival of tumor cells in the IP cavity, reminiscent of dormant cell populations. Like other dormant tumor cells, in vivo-isolated and in vitro-cultured spheroids exhibit enhanced chemoresistance and display markers of quiescence. We show that expression of mitochondrial superoxide dismutase (Sod2), which regulates mitochondrial redox balance, is significantly increased during formation of dense spheroids from a number of ovarian cancer cell lines. This is accompanied by increases in the quiescence marker p27(kip1). Importantly, siRNA and shRNA knock-down of Sod2 compromise spheroid formation and integrity, and abrogate increases in p27(kip1) expression in anchorage-independent 3D cultures. Mechanistically, it is likely that increases in Sod2 expression protect cells from build-up of mitochondrial and IP cavity-derived ROS, enhancing their survival as dormant spheroid populations. In addition, bioenergetic data suggest that Sod2 is directly involved in enhancing the mitochondrial integrity of tumor cells within spheroid aggregates. This leads to a metabolic profile that is associated with a slowing in ATP demand and an enhanced respiratory reserve capacity, with Sod2 being intricately involved in manipulating these changes. Enhanced Sod2 expression, a concomitant decrease in mitochondrial ROS and changes in mitochondrial function are likely indicators of a quiescence adaptation of tumor spheroids, necessary for survival in the IP cavity. Manipulating mitochondrial function and redox balance by effectively targeting mitochondrial antioxidant enzymes may provide novel therapeutic strategies to combat recurrence and metastatic spread of ovarian cancer.

Citation Format: LP Madhubhani Hemachandra, Usawadee Dier, Larissa M. Uusitalo, Nadine Hempel. The mitochondrial antioxidant Superoxide Dismutase 2 is necessary for ovarian cancer spheroid formation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B65.
Abstract B77: Targeting ovarian cancer promoting effects of adipocytes by metformin
Authors: Rattan Ramandeep , Tebbe Calvin , Chhina Jasdeep , Sarigiannis Kalli , Giri Shailendra , Munkarah Adnan
Published: August 2014

At the time of surgery, 80% of the ovarian cancers are found to be metastasized to the omentum, a large fat pad covering the abdominal organs. Omental metastasis is thought to be due to the promotion of homing, growth and migration of ovarian cancer cells by factors secreted by adipocytes, which include cytokines, adipokines and growth factors. These adipocytes also act as fuel depot providing for the energy needs of fast growing ovarian tumor cells. The objective of our study was to investigate if metformin can modulate or limit the adipocyte mediated tumor-promoting and migrating effects. For this, mouse preadipocyte cells were differentiated in the presence or absence of metformin and various parameters including lipid accumulation, AMPK activation and adipogenesis transcription factors were examined. Conditioned media from undifferentiated and differentiated adipocytes was used to evaluate ID-8 mouse ovarian cancer cell proliferation and migration. Conditioned media from differentiated but not from undifferentiated preadipocytes, increased ID-8 cell proliferation (p<0.05) and migration (p<0.050), which were attenuated in presence of metformin (p<0.05). Metformin treatment inhibited adipogenesis as evident by the reduction of neutral lipid accumulation. Metformin treatment increased AMP-activated protein kinase (AMPK) activity as evident from higher level of phosphorylation of AMPK and its immediate downstream target ACC (Acetyl Co Carboxylase). It also inhibited the key adipogenesis transcription factors (CEBPαa; CEBPβb, SREBP1) during the process of adipocyte diffrenetiation. A targeted Cancer Pathway Finder RT-PCR based gene array (Qiagen) revealed 21 genes that were upreguated and 2 were downregulated more than 2-fold in ID-8 cells in presence of adipocyte conditioned media, which were modulated by metformin.

Together, metformin treatment inhibited the adipocyte mediated ovarian cancer cell proliferation and migration. It also blunted the expression of cancer associated genes that were induced by adipocyte influence in cancer cells. This study suggests that metformin could be a therapeutic option for ovarian cancer as it not only targets ovarian cancer but alternatively, also modulates the environmental milieu around it.

Citation Format: Ramandeep Rattan, Calvin Tebbe, Jasdeep Chhina, Kalli Sarigiannis, Shailendra Giri, Adnan Munkarah. Targeting ovarian cancer promoting effects of adipocytes by metformin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B77.
Abstract IA12: Ovarian cancer: Time for a new paradigm
Authors: Ozols Robert F.
Published: August 2014

The majority of patients with ovarian cancer are still diagnosed with advanced stage disease as screening and early detection have not been successful. The cornerstones of therapy have been surgical cytoreduction followed by combination chemotherapy. Numerous clinical trials in the past 4 decades have evaluated various chemotherapeutic approaches and different treatment modalities. Despite this intensive research effort, standard therapy remains surgery and chemotherapy with paclitaxel plus carboplatin. Most patients will achieve a clinical complete remission but over 75% will relapse and recurrent ovarian cancer is not curable. While 5-year survival rates have improved, there has been no significant improvement in overall survival. It is not likely that significant improvements in either surgery or cytotoxic chemotherapy are forthcoming. Molecular targeted therapy is under intensive investigation. However, antiangiogenesis treatment, and PARP inhibiors ( in BRCA patients) have, as yet, only improved progression free survival without an improvement in survival. More specific molecular targets must be identified. Functional genomics, improved pre-clinical models, and a better understanding of the molecular pathogenesis of ovarian cancer perhaps hold the key to novel therapies which will increase the cure rate.

Citation Format: Robert F. Ozols. Ovarian cancer: Time for a new paradigm. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA12.
Abstract IA18: Evolutionary dynamics of high-grade serous ovarian cancer genomes across anatomic space and time
Authors: Shah Sohrab
Published: August 2014

High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to emergence of treatment-resistant sub-clones. We sought to measure the degree of genomic diversity within primary, untreated HGSC to examine the natural state of tumor evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on thirty-one spatially and temporally separated HGSC tumor specimens (six patients) including ovarian masses, distant metastases, and fallopian tube lesions. We found widespread intra-tumoral variation in mutation, copy number, and gene expression profiles, with key driver alterations in genes present in only a subset of samples (e.g. PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range: 10.2%-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology with common etiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating diversity arises at early stages of tumorigenesis. Our results reveal that HGSC exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug resistance mechanisms.

Citation Format: Sohrab Shah. Evolutionary dynamics of high-grade serous ovarian cancer genomes across anatomic space and time. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA18.
Abstract IA19: Intratumoral heterogeneity and drug response in high-grade serous carcinoma
Authors: Brenton James D.
Published: August 2014

Our understanding of the molecular basis of epithelial ovarian cancer has changed dramatically over the past 10 years. We now recognise that different histological subtypes are fundamentally different diseases, based on recent discoveries identifying the cell-of-origin and key driver mutations for high-grade serous (HGSOC) and clear cell ovarian cancers. The major challenge for the development of personalized treatment for HGSOC is the extreme genomic heterogeneity between cases and additional intratumoural heterogeneity (ITH). We have previously shown that HGSOC is characterised by ubiquitous TP53 mutation and we have proposed that early loss of TP53 may be permissive for the development of a wide range of mutator phenotypes, which may contribute to platinum resistance and ITH.

I will discuss our recent findings showing profound ITH in HGSOC and how tandem mutator phenotype may alter evolution in HGSOC. We have developed new methods to identify and quantify circulating tumour DNA in plasma samples from women with HGSOC. These methods provide highly specific biomarkers of response to treatment and can be used to track the emergence of resistant clonal populations. Application of these methods in a national study of relapsed ovarian cancer and in new clinical trials will provide critical clinical data to develop personalized treatment strategies for women with HGSOC.

Citation Format: James D. Brenton. Intratumoral heterogeneity and drug response in high-grade serous carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA19.
Abstract IA20: Analyzing the cellular basis for heterogeneity in serous ovarian carcinoma
Authors: Neel Benjamin G.
Published: August 2014

Serous ovarian cancer (SOC) is the leading cause of morbidity/mortality from gynecologic cancer. Current therapies increase survival, yet the majority of SOC patients die of their disease. The cancer stem cell (CSC) hypothesis holds that only a subset of tumor cells can initiate/maintain the tumor and might be more resistant to chemotherapy. In the CSC model, it should be possible to purify a stable population with the ability to generate transplantable tumors that recreate the heterogeneity of the initial malignancy. We previously reported that CD133 marks all TIC from several primary SOC, but in some cases, TIC activity is also found in the CD133– fraction. Also, the TIC phenotype is unstable in xenografts.

To address whether the expression of an unidentified protein marks all TIC in SOC, we performed high-throughput flow cytometry (profiling 365 cell surface markers) and combined these analyses with mass cytometry, a transformative technology allowing examination of 35 cell surface/intracellular markers on a single cell. Analysis of 40 primary SOC samples provided further evidence for inter- and intra-patient heterogeneity. Current analyses focus on functional validation of subpopulations marked by combinations of cell surface/stem cell genes.

In the course of these studies, we have generated a large cohort of SOC xenografts. Xenografts recapitulate the inter- and intra-patient heterogeneity of SOC when their gene expression and copy number profiles are compared. Indeed, they also recapitulate the patient's sensitivity to standard of care chemotherapy. These xenografts might provide a useful model l for more efficient and personalized testing of investigational drugs for SOC.

Citation Format: Benjamin G. Neel. Analyzing the cellular basis for heterogeneity in serous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA20.
Abstract IA27: Combinatorial immunotherapy using whole tumor antigen: Evidence from phase I trials
Authors: Kandalaft Lana E. , Tanyi Janos , Chiang Cheryl , Coukos George
Published: August 2014

Novel therapeutic strategies are warranted in recurrent ovarian cancer. We report two independent consecutive studies of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy. Thirty one patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from debulking surgery enrolled in two different vaccination studies. First six underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide followed by vaccination with an autologous DC preparation pulsed with freeze-thaw autologous tumor lysate while the other 25 underwent vaccination with an enhanced vaccine of autologous DCs loaded with HOCl-oxidized autologous tumor lysate administered intranodally in combination with antiangiogenesis therapy. Both studies were followed by lymphodepletion and transfer of autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T-cells, in combination with antiangiogenesis therapy and vaccination. Feasibility, safety, biological and clinical efficacy were evaluated.

Eleven patients have completed vaccination and T cell transfer to date, while twenty-three additional patients completed vaccination only. Vaccination was well tolerated and elicited tumor-specific T cell responses against various ovarian tumor antigens. Preliminary results demonstrate that patients' DCs loaded with HOCl-oxidized lysate elicited strong tumor-specific IFN-gamma; secretions and produced high levels of Th1-priming cytokines and chemokines, including IL-12. A clinical benefit of 65% correlated with the immune response with some patients experiencing prolonged progression free survival. Following lymphodepletion, adoptive transfer of vaccine-primed T-cells was well tolerated and resulted in durable reduction of T-regulatory cells and restoration of vaccine-induced antitumor immunity in patients who experienced clinical benefit. One patient exhibited a complete response at end of study and stable disease was observed in 7 out of the 11 patients who completed vaccination and T cell transfer. Data on additional patients will be presented at the meeting.

Our results suggest the use of combinatorial cellular immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of patients with recurrent ovarian cancer is promising yet warrants further investigation.

Citation Format: Lana E. Kandalaft, Janos Tanyi, Cheryl Chiang, George Coukos. Combinatorial immunotherapy using whole tumor antigen: Evidence from phase I trials. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA27.
Abstract IA7: Determinants of clinical outcome in ovarian cancer
Authors: Bowtell David D.L.
Published: August 2014

Clinical outcome for ovarian cancer has seen little improvement over the last two decades despite intensive research and clinical trial activity. Why has ovarian cancer been so intractable? Patient cohort studies that seek to unravel the determinants of clinical outcome allow a focus on molecular changes that are important to patient health, as well as providing experiments of nature that help researchers identify drivers of disease. The presentation will provide examples of somatic and germline determinants of patient outcome that must be considered when seeking biomarkers of tumor behavior. Intratumoral heterogeneity and tumor evolution during therapy will also be discussed.

Ovarian cancer has been fragmented into distinct molecular subtypes and the origins of the different histotypes revised. However, fragmentation has also resulted in unexpected convergence, involving the discovery of molecular similarities of ovarian cancer subtypes with other solid cancers. Ovarian cancer provides an excellent example of a reclassification of solid cancer that is currently underway, based on molecular properties of a tumor rather than anatomy.

Citation Format: David D.L. Bowtell. Determinants of clinical outcome in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA7.
Abstract PR04: DNA-PK inhibition resensitizes ovarian cancer cells to cisplatin in vivo and is associated with modulation of AKT pathway signaling
Authors: Cheraghchi-Bashi-Astaneh Azadeh , Sousa Camila H. De , Maginn Elaina , Dai Yan , Wasan Harpreet , Mills Gordon B. , Gabra Hani , Stronach Euan A.
Published: August 2014

Sustained proliferative signaling and resisting cell death are two fundamental hallmarks of cancer. The AKT pathway is a frequently aberrant, highly interconnected, intracellular signaling network that connects a wide variety of cell stimuli to proliferative and apoptotic cellular control. We have shown that inappropriate control of the AKT pathway underlies failure of chemotherapy in ovarian cancer and furthermore have shown that DNA-PKcs, a component of the non-homologous-end-joining DNA repair pathway, phosphorylates AKT on serine residue 473 in the nucleus of platinum resistant ovarian tumor cells, but not sensitive cells from the same patient. DNA-PK mediated AKT activation in chemoresistant cells results in inhibitory phosphorylation of the pro-apoptotic protein BAD and stabilization of the anti-apoptotic Bcl-2 protein. We report that inhibition of DNA-PK restores response to cisplatin in chemoresistant ovarian cancer cells in vivo. SKOV3 tumor xenografts were implanted subcutaneously into the flanks of Balb/c nu/nu mice and treated with 7.5mg/kg or 10mg/kg DNA-PKcs inhibitor, NU7441 daily (5days on/2 days off) for 2 weeks alone or in combination with 1mg/kg cisplatin twice weekly. DNA-PK inhibition or cisplatin alone were ineffective however in combination decreased tumor growth at 14 days by 89% (7.5mg/kg NU7441) and 90% (10mg/kg NU7441), relative to platinum only treatment. Tumor xenografts were recovered and analyzed by reverse phase protein array (RPPA). Analysis has revealed AKT pathway signaling alterations that underlie the enhanced response to cisplatin on addition of NU7441. DNA-PK inhibition is an attractive therapeutic strategy for resensitizing resistant tumors to platinum based therapy, directly linking the DNA damage caused by platinum to the pro-survival AKT pathway.

This abstract is also presented as Poster A50.

Citation Format: Azadeh Cheraghchi-Bashi-Astaneh, Camila H. De Sousa, Elaina Maginn, Yan Dai, Harpreet Wasan, Gordon B. Mills, Hani Gabra, Euan A. Stronach. DNA-PK inhibition resensitizes ovarian cancer cells to cisplatin in vivo and is associated with modulation of AKT pathway signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR04.
Integrated Analysis of Transcriptomes of Cancer Cell Lines and Patient Samples Reveals STK11/LKB1–Driven Regulation of cAMP Phosphodiesterase-4D
Authors: He Ningning , Kim Nayoung , Song Mee , Park Choa , Kim Somin , Park Eun Young , Yim Hwa Young , Kim Kyunga , Park Jong Hoon , Kim Keun Il , Zhang Fan , Mills Gordon B. , Yoon Sukjoon
Published: August 2014

The recent proliferation of data on large collections of well-characterized cancer cell lines linked to therapeutic drug responses has made it possible to identify lineage- and mutation-specific transcriptional markers that can help optimize implementation of anticancer agents. Here, we leverage these resources to systematically investigate the presence of mutation-specific transcription markers in a wide variety of cancer lineages and genotypes. Sensitivity and specificity of potential transcriptional biomarkers were simultaneously analyzed in 19 cell lineages grouped into 228 categories based on the mutational genotypes of 12 cancer-related genes. Among a total of 1,455 category-specific expression patterns, the expression of cAMP phosphodiesterase-4D (PDE4D) with 11 isoforms, one of the PDE4(A-D) subfamilies, was predicted to be regulated by a mutant form of serine/threonine kinase 11 (STK11)/liver kinase B1 (LKB1) present in lung cancer. STK11/LKB1 is the primary upstream kinase of adenine monophosphate–activated protein kinase (AMPK). Subsequently, we found that the knockdown of PDE4D gene expression inhibited proliferation of STK11-mutated lung cancer lines. Furthermore, challenge with a panel of PDE4-specific inhibitors was shown to selectively reduce the growth of STK11-mutated lung cancer lines. Thus, we show that multidimensional analysis of a well-characterized large-scale panel of cancer cell lines provides unprecedented opportunities for the identification of unexpected oncogenic mechanisms and mutation-specific drug targets. Mol Cancer Ther; 13(10); 2463–73. ©2014 AACR.
Abstract A10: In vivo models of pancreatic cancer for translational medicine.
Authors: Behrens Diana , Hallas Cora , Anders Diana , Fichtner Iduna
Published: August 2014

In Germany pancreatic cancer remains a lethal disease with only 3-8% of patients surviving 5 years after diagnosis of the tumor (2005). Reasons for this poor situation are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel therapies is the restricted availability of preclinical models with a high clinical relevance. The aim of our study was to develop well-defined in vivo xenografts derived from patient pancreatic carcinomas to address translational questions. These xenografts can be used for identification of biomarkers and cancer related pathways as well as for the evaluation of targeted therapies. The genesis of pancreatic neoplasm, the progression of tumor growth as wells as the development of resistance can be elucidated with animal models of patient-derived material. And last but not least, preclinical data can be used prospectively for efficient stratification of patients. Fifty-two patient tumors were collected from the clinical cooperation partner and transplanted immediately into immune-deficient mice. Nine out of 52 samples could be established so far as passagable pancreatic cancer xenografts (PDX), 5 were identified as inflammations of the pancreas. Interestingly, we observed 4 out of 52 as post-transplant lymphoproliferative disorders (PLTD). Twenty-nine failed to grow in mice and 5 are still under observation and potential candidates for the model library. All of the engrafted PDX are poor or moderate differentiated adenocarcinomas. Global gene expression analysis and determination of cancer associated mutations were performed from engrafted tumor models. According to clinical data we found K-ras mutations in 8 and additionally p53 mutations in 4 out of 9 PDX. Chemosensitivity was evaluated by transplanting tumor material into cohorts of immune-deficient mice. Beginning from palpable tumor sizes mice were treated with 5-fluorouracil, Irinotecan, Vincristine, Avastin, Gemcitabine and Sorafenib at optimized schedules and doses. The most efficient therapeutics were Gemcitabine and Irinotecan, whereas the growth of PDX was only marginally inhibited by Vincristine and Avastin. Patient-derived xenografts are a crucial tool for the prediction of therapy and potentially relevant for the implication into clinical decisions. We have successfully developed a panel of 9 pancreatic cancer PDX for translational research projects.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A10.

Citation Format: Diana Behrens, Cora Hallas, Diana Anders, Iduna Fichtner. In vivo models of pancreatic cancer for translational medicine. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A10.
Abstract A100: KLF8 induces breast stemness and chemoresistance via miRNAs associated with EMT.
Authors: Wang Xianhui , Lu Heng , Li Tianshu , Yu Lin , Liu Gang , Peng Xu , Zhao Jihe
Published: August 2014

The mechanisms behind the induction of stem cell properties by the epithelial to mesenchymal transition (EMT) remain largely undefined. Here we report the identification of the microRNA-146a (miR-146a) as a common target of the known EMT-inducers Krüppel-like factor 8 (KLF8) and TGF-β. Upon KLF8 overexpression or TGF-β treatment, a significant portion of the MCF-10A cells gained stem cell traits as demonstrated by an increase in the expression of CD44high/CD24low, the activity of aldehyde dehydrogenase (ALDH) and the mammosphere forming capability. Along with this change, the expression of miR-146a was highly upregulated in the cells. Consistently, we found that miR-146a was aberrantly co-overexpressed with KLF8 in a panel of invasive human breast cancer cell lines. Ectopic expression of KLF8 failed to induce the stem cell traits in the MCF-10A cells if the cells were pre-treated with miR-146a inhibitor, whereas overexpression of miR-146a in the MCF-10A cells alone was sufficient to induce the stem cell traits. Importantly, this induction of miR-146a-dependent stem traits by KLF8 was well correlated to the cell resistance to the cytotoxic effects of chemotherapeutic drugs such as paclitaxel. Co-staining and luciferase reporter analyses indicated that miR-146a targets the 3’-UTR of the Notch signaling inhibitor NUMB for translational inhibition. Overexpression of KLF8 dramatically potentiated the tumorigenecity of MCF-10A cells expressing the H-Ras oncogene, which was accompanied by a loss of NUMB expression in the tumors. Taken together, this study identifies a novel role and mechanism for KLF8 in inducing pro-tumorigenic mammary stemness and chemoresistance via miR-146a potentially by activating Notch signaling. This mechanism could be exploited as a therapeutic target against drug resisting breast cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A100.

Citation Format: Xianhui Wang, Heng Lu, Tianshu Li, Lin Yu, Gang Liu, Xu Peng, Jihe Zhao. KLF8 induces breast stemness and chemoresistance via miRNAs associated with EMT. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A100.
Abstract A107: In vivo screen of regorafenib in a panel of low passage START-PDX colorectal models: Working to identify a sensitive patient subpopulation.
Authors: Wick Michael J. , Gamez Lizette , Diaz Armando , Vaught Teresa L. , Tolcher Anthony W. , Rasco Drew , Patnaik Amita , Smith Lon , Drengler Ron , Papadopoulos Kyriakos P.
Published: August 2014

Background: Regorafenib is a multi-tyrosine kinase recently approved for unresectable and drug refractory gastrointestinal stromal tumors (GIST) and treatment-refractory colorectal cancers regardless of Ras status. While a subset of colorectal cancer patients have reported benefit from regorafenib therapy, the majority does not benefit and attempts to identify a subpopulation of sensitive colorectal patients have not been successful. To better understand which colorectal cancer patients may respond to regorafenib we screened a panel of low passage colorectal models from the START patient-derived xenograft (PDX) platform and compared results to model characteristics.

Methods: PDX colorectal models were established in athymic nude mice from primary or metastatic patient tissue or fluid and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model DNA was extracted and subjected to mutation profiling and growth factor receptor expression was interrogated using immunohistochemistry. Drug sensitivity studies were performed evaluating regorafenib at 25-30 mg/kg once daily for at least twenty-one days. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion.

Results: To date forty colorectal START-PDX models have been screened against regorafenib. Three models (ST201, ST238 and ST800) reported tumor regressions or partial responses even with activating K-Ras or B-Raf mutations present. An additional 30% of evaluated models reported tumor stasis (T/C= 0-10%) and remaining models demonstrated varying degrees of sensitivity or resistance to regorafenib. High expression of EGFR was reported in two of the most sensitive models and was reduced in the models resistant to regorafenib.

Conclusion: We have screened preclinical activity of regorafenib towards a panel of colorectal START-PDX models and identified sensitive and resistant models. Additional genomic and characterization studies are underway to better understand activity of regorafenib with the goal of identifying a sensitive patient subpopulation.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A107.

Citation Format: Michael J. Wick, Lizette Gamez, Armando Diaz, Teresa L. Vaught, Anthony W. Tolcher, Drew Rasco, Amita Patnaik, Lon Smith, Ron Drengler, Kyriakos P. Papadopoulos. In vivo screen of regorafenib in a panel of low passage START-PDX colorectal models: Working to identify a sensitive patient subpopulation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A107.
Abstract A144: Inhibition of 2-HG production in IDH mutant xenograft models.
Authors: Slocum Kelly L. , Downall Julia , Gould Ty , Zafari Mohammad , Dodd Stephanie , Firestone Brant , Pagliarini Ray , Levell Julian
Published: August 2014

Isocitrate dehydrogenase 1 and 2 (IDH1/2) oxidize isocitrateto α-ketoglutarate (α-KG), a cofactor needed for the function of over 100 enzymes1. Heterozygous mutations of IDH1/2 occur in a variety of tumor types including glioma, acute myeloid leukemia (AML), cholangiosarcoma, chondrosarcoma and melanoma2. Mutant IDH is crippled for wild-type function but gains the ability to convert α-KG to 2-hydroxyglutarate (2-HG), a believed “oncometabolite” that may alter cell biology through, in part, changes in global histone and DNA methylation 1,3,4.

However, the dearth of in vivo models dependent upon mutant IDH has made understanding the biological relevance of IDH mutations and 2-HG production in cancer challenging. To better understand the role that IDH mutations play in oncogenesis, and the potential effects of inhibition of these mutations, we introduced a heterozygous point mutation of IDH1 (R132H) into the endogenous locus of the HCT116 colon carcinoma cell line. When compared with the parental IDH1 wild-type HCT116 model, HCT116 IDH1R132H/+ xenograft tumors produce significantly higher levels of 2-HG. In addition, we have identified a patient-derived melanoma model, HMEX2838, which harbors an endogenous IDH1R132C/+ mutation and expresses high levels of 2-HG.

To better explore the biology of mutant IDH inhibition, we developed a mutant-selective IDH inhibitor with favorable in vivo properties. Using this compound we demonstrate that 2-HG production is strongly inhibited in both the engineered HCT116 IDH1R132H/+ xenograft model and the endogenously mutant HMEX2838 patient-derived xenograft model. The data we have generated suggests that this inhibition of 2-HG directly correlates with the area under the curve (AUC) of free-drug above the HCT116 IDH1R132H/+ cellular IC50, as determined in vitro. Using these models and our IDH inhibitors we hope to gain a better understanding of how IDH mutations contribute to cancer and how inhibition of the mutant enzyme could benefit patients.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A144.

Citation Format: Kelly L. Slocum, Julia Downall, Ty Gould, Mohammad Zafari, Stephanie Dodd, Brant Firestone, Ray Pagliarini, Julian Levell. Inhibition of 2-HG production in IDH mutant xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A144.
Abstract A164: Targeting PDK1 in breast cancer: Kinase-dependent regulation of tumor growth and kinase-independent regulation of cell migration and invasion.
Authors: Gagliardi Paolo Armando , di Blasio Laura , Seano Giorgio , Sessa Roberto , Puliafito Alberto , Bussolino Federico , Primo Luca
Published: August 2014

3-Phosphoinositide Dependent Kinase 1 (PDK1) is the kinase that phosphorylates the activation loop of several protein kinases such as Akt, PKC or SGK. In breast carcinomas PDK1 was found frequently overexpressed and this correlates with a more aggressive phenotype. This study is aimed to understand the role of PDK1 in breast tumor progression and the molecular mechanisms involved. Using stable gene silencing and kinase inhibitors we studied PDK1 role in different in vitro and in vivo assays including anchorage independent growth, anoikis, xenografts, migration and invasion assays. In our study we found that PDK1 promotes resistance to anoikis, anchorage independent growth and tumor growth in breast cancer cell lines through the phosphorylation of its substrates, since the kinase-dead mutant abrogates all the PDK1-mediated effects. For this reasons either PDK1 gene silencing or its chemical kinase inhibition are sufficient to promote anoikis, to reduce anchorage independent growth and tumor growth. We also found that PDK1 silencing is able to reduce, while its overexpression to increase cell migration and invasion in mammary cells. Surprisingly, the regulation of cell migration is operated in a kinase independent manner, in fact PDK1 kinase inhibitor failed to recapitulate the effects of gene silencing in cell migration. The molecular mechanism, underlying this unconventional kinase-independent regulation of cell migration involves the binding of PDK1 to Myotonic Dystrophy kinase-related Cdc42-binding Kinase α (MRCKα). This binding increases MRCKα kinase activity on Myosin Light Chain-2 (MLC2) and on myosin phosphatase target subunit 1 (MyPT1). Both these phosphorylations promote increase of non-muscular myosin activity, which is required to pull forward the cell body during directional migration and invasion. In summary, our findings unveil that PDK1 regulates tumor growth, survival in absence of adhesion, resistance to anoikis, but also migration and invasion of breast cancer cells by two different mechanisms. These findings should be taken in account for a rational targeting of PDK1 in cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A164.

Citation Format: Paolo Armando Gagliardi, Laura di Blasio, Giorgio Seano, Roberto Sessa, Alberto Puliafito, Federico Bussolino, Luca Primo. Targeting PDK1 in breast cancer: Kinase-dependent regulation of tumor growth and kinase-independent regulation of cell migration and invasion. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A164.
Abstract A172: PM060184 is a new tubulin binding agent that induces strong antitumor activity in xenografted tumors.
Authors: Guillén Maria José , Cataluña Oscar , Palomares Mandy , Aviles Pablo M.
Published: August 2014

PM060184 is a new synthetic tubulin-binding agent, originally isolated from the marine sponge Lithoplocamia lithistoides. PM060184 has demonstrated to display a very strong in vitro antitumor activity against a broad panel of tumor cell lines. The in vivo evaluation for antitumor activity of PM060184 was accomplished by using a panel of five human-derived xenografted tumors: colon (HCT-116), gastric (HGC-27), NSCLC (H460), prostate (22RV1) and renal (Caki-1). Briefly, 4 to 6 week-old athymic nu/nu mice were subcutaneously implanted into their right flank with 0.050 mL of a mixture (50:50; v/v) of cell suspension and Matrigel. When tumors reached ca. 150-200 mm3, tumor bearing animals (N=10/group) were randomly allocated into the following treatment groups: i) Placebo; ii) PM060184 (8 mg/kg); and, iii) PM060184 (16 mg/kg). Treatments were intravenously administered once per week for 3 consecutive weeks (days 0, 7 and 14). Tumor dimensions were recorded 3 times per week starting from the first day of treatment (Day 0). Tumor volume was calculated using the equation (a·b2)/2, where a and b were the longest and shortest diameters, respectively. Antitumor effect was calculated by using ΔT/ΔC, defined as the percentage of the change in tumor volume for each treated (T) and placebo (C) group. Complete tumor regressions (CR) were monitorized during the experimental period. Results are summarized in Table 1. PM060184 induced very strong antitumor effect in all the tumors evaluated and was able to induce complete tumor regressions in HCT-116, HGC-27 and 22RV1 xenografted tumors. Also, PM060184 demonstrated its capability to in vivo induce extensive necrotic areas in H460 and 22RV1 tumors. Taken together, the results presented in this work support the development of PM060184 as an anticancer agent in the clinic.

Table 1.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A172.

Citation Format: Maria José Guillén, Oscar Cataluña, Mandy Palomares, Pablo M. Aviles. PM060184 is a new tubulin binding agent that induces strong antitumor activity in xenografted tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A172.
Abstract A185: Menin functions as a gatekeeper of the proliferative MAPK arm of K-Ras signaling.
Authors: Chamberlain Chester , German Michael
Published: August 2014

Activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas, but have never been found in pancreatic endocrine tumors, which more commonly inactivate tumor suppressors such as MEN1, the gene mutated in the autosomal dominant cancer syndrome Multiple Endocrine Neoplasia type 1, showing that outcomes of K-Ras signaling depend on cellular context. We have shown that K-Ras does not promote but instead strongly suppresses endocrine cell growth and that this paradoxical activity derives from the Men1 gene product Menin. We have proposed a model in which K-Ras activates both the pro-proliferative MAPK pathway, and the anti-proliferative RASSF1 pathway. In tissues susceptible to MEN1 gene mutation, Menin normally prevents the MAPK effector pathway from driving proliferation while leaving the inhibitory RASSF1 effector pathway intact. In this model, loss of Menin causes proliferation due to removal of the block in MAPK-driven proliferation downstream of K-Ras, while loss of K-Ras signaling increases proliferation by decreasing the unopposed RASSF1 activity in susceptible cells. To test this model, we assessed the effect of various inhibitors of the K-Ras pathway on beta cell proliferation in cultured mouse islets. We found that treatment with a combination of farnesyl- and geranyl-geranyl-transferase inhibitors, which block K-Ras signaling, stimulated beta cell proliferation; but blocking MAPK signaling with an inhibitor of Mek1/2 had no effect on proliferation rate. However, in beta-cells with reduced Men1 gene dosage, both K-Ras and Mek1/2 inhibition reduced the increased basal replication rate, consistent with our model that Menin specifically blocks pro-proliferative outputs of the MAPK arm of K-Ras signaling. Remarkably, complete loss of Men1 created a synthetic lethal interaction with Mek1/2 inhibition. Our study suggests that inhibitors of the Raf/MEK/ERK pathway offer a rational therapy for tumors with germline or somatic MEN1 mutations.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A185.

Citation Format: Chester Chamberlain, Michael German. Menin functions as a gatekeeper of the proliferative MAPK arm of K-Ras signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A185.