47 results on '"Sara J. Adair"'
Search Results
2. Drp1 Promotes KRas-Driven Metabolic Changes to Drive Pancreatic Tumor Growth
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Sarbajeet Nagdas, Jennifer A. Kashatus, Aldo Nascimento, Syed S. Hussain, Riley E. Trainor, Sarah R. Pollock, Sara J. Adair, Alex D. Michaels, Hiromi Sesaki, Edward B. Stelow, Todd W. Bauer, and David F. Kashatus
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Biology (General) ,QH301-705.5 - Abstract
Summary: Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRasG12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms. : Nagdas et al. find that the mitochondrial fission GTPase Drp1 is required for KRas-driven transformation and pancreatic tumor growth. The inhibition of Drp1 in cells expressing oncogenic KRas leads to impaired glycolytic flux and the eventual loss of mitochondrial metabolic function. Keywords: Drp1, mitochondria, metabolism, KRas, pancreatic cancer
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- 2019
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3. Co-Treatment with Panitumumab and Trastuzumab Augments Response to the MEK Inhibitor Trametinib in a Patient-Derived Xenograft Model of Pancreatic Cancer
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James M. Lindberg, Timothy E. Newhook, Sara J. Adair, Dustin M. Walters, Alison J. Kim, Edward B. Stelow, J. Thomas Parsons, and Todd W. Bauer
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and epidermal growth factor receptor (EGFR) family signaling are drivers of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated that combinatorial treatment of PDAC xenografts with the mitogen-activated protein kinase–extracellular-signal-regulated kinase (ERK) kinase1/2 (MEK1/2) inhibitor trametinib and the dual EGFR/human epidermal growth factor receptor 2 (HER2) inhibitor lapatinib provided more effective inhibition than either treatment alone. In this study, we have used the therapeutic antibodies, panitumumab (specific for EGFR) and trastuzumab (specific for HER2), to probe the role of EGFR and HER2 signaling in the proliferation of patient-derived xenograft (PDX) tumors. We show that dual anti-EGFR and anti-HER2 therapy significantly augmented the growth inhibitory effects of the MEK1/2 inhibitor trametinib in three different PDX tumors. While significant growth inhibition was observed in both KRAS mutant xenograft groups receiving trametinib and dual antibody therapy (tumors 366 and 608), tumor regression was observed in the KRAS wild-type xenografts (tumor 738) treated in the same manner. Dual antibody therapy in conjunction with trametinib was equally or more effective at inhibiting tumor growth and with lower apparent toxicity than trametinib plus lapatinib. Together, these studies provide further support for a role for EGFR and HER2 in pancreatic cancer proliferation and underscore the importance of therapeutic intervention in both the KRAS–rapidly accelerated fibrosarcoma kinase (RAF)–MEK–ERK and EGFR-HER2 pathways to achieve maximal therapeutic efficacy in patients.
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- 2014
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4. Inhibition of the Growth of Patient-Derived Pancreatic Cancer Xenografts with the MEK Inhibitor Trametinib Is Augmented by Combined Treatment with the Epidermal Growth Factor Receptor/HER2 Inhibitor Lapatinib
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Dustin M. Walters, James M. Lindberg, Sara J. Adair, Timothy E. Newhook, Catharine R. Cowan, Jayme B. Stokes, Cheryl A. Borgman, Edward B. Stelow, Bryce T. Lowrey, Maria E. Chopivsky, Tona M. Gilmer, John T. Parsons, and Todd W. Bauer
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.
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- 2013
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5. Cancer-oocyte SAS1B protein is expressed at the cell surface of multiple solid tumors and targeted with antibody-drug conjugates
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Craig L Slingluff, Walter C Olson, Christine A Tran, Arabinda Mandal, Jagathpala Shetty, Mriganka Mandal, Bhupal Ban, Eusebio S Pires, Sara J Adair, Todd W Bauer, and John C Herr
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Background Sperm acrosomal SLLP1 binding (SAS1B) protein is found in oocytes, which is necessary for sperm-oocyte interaction, and also in uterine and pancreatic cancers. Anti-SAS1B antibody-drug conjugates (ADCs) arrested growth in these cancers. However, SAS1B expression in cancers and normal tissues has not been characterized. We hypothesized that SAS1B is expressed on the surface of other common solid cancer cells, but not on normal tissue cells, and might be selectively targeted therapeutically.Methods SAS1B expression in human normal and cancer tissues was determined by immunohistochemistry, and complementary DNA (cDNA) libraries were employed to PCR amplify human SAS1B and its transcripts. Monoclonal antibodies (mAbs) to human SAS1B were generated using mouse hybridomas. SAS1B deletion constructs were developed to map SAS1B’s epitope, enabling the creation of a blocking peptide. Indirect immunofluorescence (IIF) of human transfected normal and cancer cells was performed to assess SAS1B expression. SAS1B intracellular versus surface expression in normal and tumor tissues was evaluated by flow cytometry after staining with anti-SAS1B mAb, with specificity confirmed with the blocking peptide. Human cancer lines were treated with increasing mAb and ADC concentrations. ATP was quantitated as a measure of cell viability.Results SAS1B expression was identified in a subset of human cancers and the cytoplasm of pancreatic islet cells. Two new SAS1B splice variants were deduced. Monoclonal antibodies were generated to SAS1B splice variant A. The epitope for mAbs SB2 and SB5 is between SAS1B amino acids 32–39. IIF demonstrated intracellular SAS1B expression in transfected kidney cells and on the cell surface of squamous cell lung carcinoma. Flow cytometry demonstrated intracellular SAS1B expression in all tumors and some normal cells. However, surface expression of SAS1B was identified only on cancer cells. SB2 ADC mediated dose-dependent cytotoxic killing of multiple human cancer lines.Conclusion SAS1B is a novel cancer-oocyte antigen with cell surface expression restricted to cancer cells. In vitro, it is an effective target for antibody-mediated cancer cell lysis. These findings support further exploration of SAS1B as a potential therapeutic cancer target in multiple human cancers, either with ADC or as a chimeric antigen receptor-T (CAR-T) cell target.
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- 2024
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6. Data from Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment
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Todd W. Bauer, J. Thomas Parsons, Edward B. Stelow, Rosa F. Hwang, Amy H. Bouton, Keena S. Thomas, Bryce Lowrey, E. Dan Hershey, Robert W. Tilghman, Dustin M. Walters, Jill K. Slack-Davis, Sara J. Adair, and Jayme B. Stokes
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Current therapies for pancreatic ductal adenocarcinoma (PDA) target individual tumor cells. Focal adhesion kinase (FAK) is activated in PDA, and levels are inversely associated with survival. We investigated the effects of PF-562,271 (a small-molecule inhibitor of FAK/PYK2) on (i) in vitro migration, invasion, and proliferation; (ii) tumor proliferation, invasion, and metastasis in a murine model; and (iii) stromal cell composition in the PDA microenvironment. Migration assays were conducted to assess tumor and stromal cell migration in response to cellular factors, collagen, and the effects of PF-562,271. An orthotopic murine model was used to assess the effects of PF-562,271 on tumor growth, invasion, and metastasis. Proliferation assays measured PF-562,271 effects on in vitro growth. Immunohistochemistry was used to examine the effects of FAK inhibition on the cellular composition of the tumor microenvironment. FAK and PYK2 were activated and expressed in patient-derived PDA tumors, stromal components, and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK (phospho-FAK or Y397) in a dose-dependent manner. PF-562,271 inhibited migration of tumor cells, cancer-associated fibroblasts, and macrophages. Treatment of mice with PF-562,271 resulted in reduced tumor growth, invasion, and metastases. PF-562,271 had no effect on tumor necrosis, angiogenesis, or apoptosis, but it did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts than control or gemcitabine. These data support a role for FAK in PDA and suggest that inhibitors of FAK may contribute to efficacious treatment of patients with PDA. Mol Cancer Ther; 10(11); 2135–45. ©2011 AACR.
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- 2023
7. supplementary Figure Legends 1-2 from Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment
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Todd W. Bauer, J. Thomas Parsons, Edward B. Stelow, Rosa F. Hwang, Amy H. Bouton, Keena S. Thomas, Bryce Lowrey, E. Dan Hershey, Robert W. Tilghman, Dustin M. Walters, Jill K. Slack-Davis, Sara J. Adair, and Jayme B. Stokes
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PDF file - 72K
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- 2023
8. Supplementary Figure 1 from Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment
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Todd W. Bauer, J. Thomas Parsons, Edward B. Stelow, Rosa F. Hwang, Amy H. Bouton, Keena S. Thomas, Bryce Lowrey, E. Dan Hershey, Robert W. Tilghman, Dustin M. Walters, Jill K. Slack-Davis, Sara J. Adair, and Jayme B. Stokes
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PDF file - 48K
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- 2023
9. Supplementary Figure 2 from Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment
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Todd W. Bauer, J. Thomas Parsons, Edward B. Stelow, Rosa F. Hwang, Amy H. Bouton, Keena S. Thomas, Bryce Lowrey, E. Dan Hershey, Robert W. Tilghman, Dustin M. Walters, Jill K. Slack-Davis, Sara J. Adair, and Jayme B. Stokes
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PDF file - 85K
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- 2023
10. Supplemental legend from CD47 Blockade as an Adjuvant Immunotherapy for Resectable Pancreatic Cancer
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Todd W. Bauer, J. Thomas Parsons, Kodi S. Ravichandran, Craig L. Slingluff, Timothy N. J. Bullock, Jesse B. Persily, Matthew G. Mullen, Sarbajeet Nagdas, Bernadette J. Goudreau, Sho Morioka, Sara J. Adair, Timothy E. Newhook, and Alex D. Michaels
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Supplemental legend
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- 2023
11. Data from CD47 Blockade as an Adjuvant Immunotherapy for Resectable Pancreatic Cancer
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Todd W. Bauer, J. Thomas Parsons, Kodi S. Ravichandran, Craig L. Slingluff, Timothy N. J. Bullock, Jesse B. Persily, Matthew G. Mullen, Sarbajeet Nagdas, Bernadette J. Goudreau, Sho Morioka, Sara J. Adair, Timothy E. Newhook, and Alex D. Michaels
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Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival.Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 (“don't eat me signal”) expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival.Results: In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival.Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415–25. ©2017 AACR.
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- 2023
12. Automated biophysical classification of apoptotic pancreatic cancer cell subpopulations by using machine learning approaches with impedance cytometry
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Carlos Honrado, Armita Salahi, Sara J. Adair, John H. Moore, Todd W. Bauer, and Nathan S. Swami
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Machine Learning ,Pancreatic Neoplasms ,Electric Impedance ,Tumor Microenvironment ,Biomedical Engineering ,Humans ,Apoptosis ,Bioengineering ,General Chemistry ,Flow Cytometry ,Biochemistry - Abstract
Unrestricted cell death can lead to an immunosuppressive tumor microenvironment, with dysregulated apoptotic signaling that causes resistance of pancreatic cancer cells to cytotoxic therapies. Hence, modulating cell death by distinguishing the progression of subpopulations under drug treatment from viable towards early apoptotic, late apoptotic, and necrotic states is of interest. While flow cytometry after fluorescent staining can monitor apoptosis with single-cell sensitivity, the background of non-viable cells within non-immortalized pancreatic tumors from xenografts can confound distinction of the intensity of each apoptotic state. Based on single-cell impedance cytometry of drug-treated pancreatic cancer cells that are obtained from tumor xenografts with differing levels of gemcitabine sensitivity, we identify the biophysical metrics that can distinguish and quantify cellular subpopulations at the early apoptotic
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- 2022
13. A histone methylation-MAPK signaling axis drives durable epithelial-mesenchymal transition in hypoxic pancreas cancer
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Brooke A. Brown, Paul J. Myers, Sara J. Adair, Jason R. Pitarresi, Shiv K. Sah-Teli, William S. Hart, Michelle Barbeau, Kelsey Leong, Nicholas Seyler, William Kane, Kyoung Eun Lee, Edward Stelow, M. Celeste Simon, Peppi Koivunen, Todd W. Bauer, Ben Z. Stanger, and Matthew J. Lazzara
- Abstract
Here, we show that hypoxia drives especially long-lasting epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) primarily through a positive-feedback histone methylation-MAPK signaling axis. We find that neoplastic PDAC cells preferentially undergo EMT in hypoxic tumor regions in multiple model systems and that hypoxia drives a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, can last for weeks. We further demonstrate that hypoxia reduces histone demethylase KDM2A activity, suppresses PP2 family phosphatase expression, and activates MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors play only a supporting role. This mechanism can be antagonizedin vivoby combinations of MAPK inhibitors that may be effective in multi-drug therapies designed to target EMT.
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- 2022
14. Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells
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Anindya Dutta, Mazhar Adli, Cem Kuscu, Denis Liu, Xiaolong Wei, Alaa Hamdi Habieb, Yusuf Mert Demirlenk, Sara J. Adair, Patrick Edward O'Hara, Todd W. Bauer, Jiekun Yang, Ebru Yilmaz, Kyung Yong Lee, and William J. Kane
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Protein-Arginine N-Methyltransferases ,endocrine system diseases ,Cell Survival ,DNA damage ,DNA repair ,Druggability ,Mice, Nude ,Antineoplastic Agents ,Synthetic lethality ,Deoxycytidine ,Gene Knockout Techniques ,Drug Development ,In vivo ,Cell Line, Tumor ,Pancreatic cancer ,medicine ,Animals ,Humans ,CRISPR ,Replication protein A ,Multidisciplinary ,business.industry ,Protein arginine methyltransferase 5 ,Cancer ,Biological Sciences ,medicine.disease ,Xenograft Model Antitumor Assays ,Gemcitabine ,Pancreatic Neoplasms ,Cancer research ,CRISPR-Cas Systems ,business ,medicine.drug - Abstract
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancer to treat. Due to the asymptomatic nature of the disease and ineffective drug treatment modalities, the survival rate of PDAC patients remains one of the lowest. The recurrent genetic alterations in PDAC are yet to be targeted; therefore, identifying effective therapeutic combinations is desperately needed. Here, we performed anin vivoCRISPR screening in a clinically relevant patient-derived xenograft (PDX) model system to identify synergistic drug combinations for PDAC treatment. Our approach revealed protein arginine methyltransferase gene 5 (PRMT5) as a promising druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to gemcitabine. Genetic and pharmacological inhibition results indicate that of PRMT5 depletion results in synergistic cytotoxicity with Gem due to depleted replication protein A (RPA) levels and an impaired non-homology end joining (NHEJ) DNA repair. Thus, the novel combination creates conditional lethality through the accumulation of excessive DNA damage and cell death, bothin vitroandin vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is an effective approach in identifying synthetic lethal drug combinations for cancer treatment.STATEMENT of SIGNIFICANCEIdentify synergistic drug combinations for PDAC is a significant unmet need. Through CRISPR screening, we discovered and validated that PRMT5 depletion creates synergistic vulnerability of PDAC cells to gemcitabine. Mechanistically, the combination impairs DNA repair, synergistic accumulation of DNA damage and cell deathin vitroandin vivo.
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- 2020
15. Electrophysiology-based stratification of pancreatic tumorigenicity by label-free single-cell impedance cytometry
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Walter Varhue, John H. Moore, Edik M. Blais, Sarbajeet Nagdas, Sara J. Adair, John S. McGrath, Armita Salahi, Nathan S. Swami, Carlos Honrado, Todd W. Bauer, Bernadette J. Goudreau, and Vahid Farmehini
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Cell type ,Microfluidics ,Cell ,02 engineering and technology ,Adenocarcinoma ,01 natural sciences ,Biochemistry ,Article ,Analytical Chemistry ,Proto-Oncogene Proteins p21(ras) ,Mice ,Cell Line, Tumor ,Electric Impedance ,medicine ,Animals ,Humans ,Environmental Chemistry ,Pancreas ,Gene ,Spectroscopy ,Genetic heterogeneity ,Chemistry ,010401 analytical chemistry ,021001 nanoscience & nanotechnology ,Phenotype ,0104 chemical sciences ,Electrophysiology ,Gene Expression Regulation, Neoplastic ,medicine.anatomical_structure ,Liver ,Cytoplasm ,Cancer research ,Heterografts ,Single-Cell Analysis ,0210 nano-technology ,Cytometry ,Carcinoma, Pancreatic Ductal - Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer lacking specific biomarkers that can be correlated to disease onset, promotion and progression. To assess whether tumor cell electrophysiology may serve as a marker for PDAC tumorigenicity, we use multi-frequency impedance cytometry at high throughput (∼350 cells/s) to measure the electrical phenotype of single PDAC tumor cells from xenografts, which are derived from primary pancreatic tumors versus those from liver metastases of different patients. A novel phase contrast metric based on variations in the high and low frequency impedance phase responses that is related to electrophysiology of the cell interior is found to be systematically altered as a function of tumorigenicity. PDAC cells of higher tumorigenicity exhibited lowered interior conductivity and enhanced permittivity, which is validated by the dielectrophoresis on the respective cell types. Using genetic analysis, we suggest the role of dysregulated Na+ transport and removal of Ca2+ ions from the cytoplasm on key oncogenic KRAS-driven processes that may be responsible for lowering of the interior cell conductivity. We envision that impedance cytometry can serve as a tool to quantify phenotypic heterogeneity for rapidly stratifying tumorigenicity. It can also aid in protocols for dielectrophoretic isolation of cells with a particular phenotype for prognostic studies on patient survival and to tailor therapy selection to specific patients.
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- 2020
16. Abstract C054: Hypoxia promotes a durable epithelial-mesenchymal transition in pancreas cancer through a histone methylation-MAPK signaling axis
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Brooke A. Brown, Paul J. Myers, Sara J. Adair, Jason R. Pitarresi, Shiv Sah Teli, Peppi Karppinen, Ben Z. Stanger, Todd W. Bauer, and Matthew J. Lazzara
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Cancer Research ,Oncology - Abstract
Pancreatic ductal adenocarcinoma (PDAC) tumors are poorly vascularized and exhibit regions of hypoxia. Here, we demonstrate that this feature of the tumor microenvironment promotes epithelial-mesenchymal transition (EMT), which occurs early in PDAC and drives chemoresistance, and we identify the underlying signaling mechanism. Analysis of publicly-available human transcriptomics and proteomics demonstrated that PDAC cells or tumors enriched in mesenchymal markers were also enriched in markers of hypoxia or HIF activity. Furthermore, in lineage-traced autochthonous, orthotopic patient-derived xenograft, and orthotopic or subcutaneous implanted cell line models of PDAC, hypoxic tumor tissue regions were enriched for neoplastic cells that had undergone EMT. In cell culture experiments, PDAC cells from human and mouse tumors exhibited an ability to undergo EMT in response to 1% O2, with loss of membranous E-cadherin, increased vimentin protein expression, and transcriptional changes indicative of both hypoxia and EMT. Moreover, EMT in response to hypoxia was substantially more persistent than that observed in response to growth factors, and a hypoxia fate mapping system revealed that once-hypoxic cells could retain mesenchymal characteristics outside hypoxic tumor regions. To understand the mechanism for EMT in response to low oxygen tension, we constructed a multivariable linear regression model of the dependence of the hypoxic gene signature on different gene sets in PDAC ductal cells, which identified MAPK signaling as the most important feature. Consistent with the model inference, in both in vitro and in vivo settings, hypoxic cells showing evidence of EMT displayed elevated MAPK signaling. We further demonstrated that MAPK activation in hypoxia was potentiated by suppressed activity of a histone demethylase and concomitant loss of protein phosphatase expression, which reinforced the mechanism by stabilizing the expression of a histone methyltransferase. Thus, this study identifies a tumor microenvironment-initiated mechanism leading to EMT and nominates several potential drug targets whose antagonism may promote PDAC chemoresponse. Citation Format: Brooke A. Brown, Paul J. Myers, Sara J. Adair, Jason R. Pitarresi, Shiv Sah Teli, Peppi Karppinen, Ben Z. Stanger, Todd W. Bauer, Matthew J. Lazzara. Hypoxia promotes a durable epithelial-mesenchymal transition in pancreas cancer through a histone methylation-MAPK signaling axis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C054.
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- 2022
17. ISL2 is a putative tumor suppressor whose epigenetic silencing reprograms the metabolism of pancreatic cancer
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Harun Ozturk, Harun Cingoz, Turan Tufan, Jiekun Yang, Sara J. Adair, Krishna Seshu Tummala, Cem Kuscu, Meric Kinali, Gamze Comertpay, Sarbajeet Nagdas, Bernadette J. Goudreau, Husnu Umit Luleyap, Yagmur Bingul, Timothy B. Ware, William L. Hwang, Ku-lung Hsu, David F. Kashatus, David T. Ting, Navdeep S. Chandel, Nabeel Bardeesy, Todd W. Bauer, and Mazhar Adli
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LIM-Homeodomain Proteins ,Nerve Tissue Proteins ,Cell Biology ,General Biochemistry, Genetics and Molecular Biology ,Epigenesis, Genetic ,Pancreatic Neoplasms ,Cell Line, Tumor ,Tumor Microenvironment ,Humans ,Genes, Tumor Suppressor ,Molecular Biology ,Developmental Biology ,Carcinoma, Pancreatic Ductal ,Transcription Factors - Abstract
Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.
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- 2021
18. Drp1 Promotes KRas-Driven Metabolic Changes to Drive Pancreatic Tumor Growth
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Sara J. Adair, Edward B. Stelow, Sarbajeet Nagdas, Hiromi Sesaki, Todd W. Bauer, Alex D. Michaels, Syed Saad Hussain, Aldo Nascimento, David F. Kashatus, Sarah R. Pollock, Jennifer A. Kashatus, and Riley E. Trainor
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0301 basic medicine ,Dynamins ,endocrine system ,Apoptosis ,Mitochondrion ,medicine.disease_cause ,Mitochondrial Dynamics ,General Biochemistry, Genetics and Molecular Biology ,Article ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Pancreatic tumor ,Pancreatic cancer ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,Glycolysis ,Phosphorylation ,lcsh:QH301-705.5 ,Cell Proliferation ,Mice, Knockout ,Chemistry ,medicine.disease ,Xenograft Model Antitumor Assays ,Cell biology ,Mitochondria ,Gene Expression Regulation, Neoplastic ,Pancreatic Neoplasms ,030104 developmental biology ,lcsh:Biology (General) ,Mitochondrial fission ,KRAS ,Flux (metabolism) ,030217 neurology & neurosurgery ,Homeostasis - Abstract
SUMMARY Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRasG12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms., In Brief Nagdas et al. find that the mitochondrial fission GTPase Drp1 is required for KRas-driven transformation and pancreatic tumor growth. The inhibition of Drp1 in cells expressing oncogenic KRas leads to impaired glycolytic flux and the eventual loss of mitochondrial metabolic function., Graphical Abstract
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- 2019
19. ISL2 is an epigenetically silenced tumor suppressor and regulator of metabolism in pancreatic cancer
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Todd W. Bauer, Turan Tufan, Mazhar Adli, Ku-Lung Hsu, Nabeel Bardeesy, Sarbajeet Nagdas, Husnu Umit Luleyap, Sara J. Adair, Krishna S. Tummala, Dave F. Kashatus, Cem Kuscu, Bernadette J. Goudreau, Jiekun Yang, Gamze Cömertpay, and Harun Cingoz
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Biology ,medicine.disease ,medicine.disease_cause ,law.invention ,Downregulation and upregulation ,law ,Pancreatic cancer ,DNA methylation ,Gene expression ,medicine ,Cancer research ,Suppressor ,KRAS ,Epigenetics ,Transcription factor - Abstract
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers. Uncovering mechanisms responsible for the heterogeneous clinical features of this disease is an essential step toward developing improved and more specific therapeutic approaches. Here, we sought to identify transcriptional regulators of aggressive PDAC growth through in vivo CRISPR screening of epigenetic and transcription factors in an orthotopic model. We identified the ISL LIM homeobox 2 (ISL2) gene as a tumor suppressor whose depletion enhances the proliferation of human PDAC cells in vitro and in vivo and cooperates with activated KRAS to initiate PDAC in a murine model. Conversely, the upregulation of ISL2 expression through CRISPR-mediated locus-specific epigenetic editing results in reduced cell proliferation. Importantly, ISL2 is epigenetically silenced through DNA methylation in ~60% of PDAC tumors, which correlates with poor patient outcome. Functional studies showed that ISL2 loss rewires metabolic gene expression, and consequently potentiates oxidative phosphorylation while reducing glycolysis. This metabolic shift creates selective vulnerability to small molecule inhibitors of mitochondrial respiration and fatty acid oxidation. Collectively, these findings reveal ISL2 as a novel tumor suppressor whose inactivation drives metabolic reprogramming in an aggressive PDAC subset and point to potential therapeutic vulnerabilities in these tumors.
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- 2020
20. CRISPR knockout screening identifies combinatorial drug targets in pancreatic cancer and models cellular drug response
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Matthew G. Mullen, Alex D. Michaels, Natasha Lopes Fischer, Mazhar Adli, P. Todd Stukenberg, Sara J. Adair, Karol Szlachta, Todd W. Bauer, Limin Liu, Stephen Shang, Turan Tufan, Edward B. Stelow, Prasad Trivedi, J. Thomas Parsons, Cem Kuscu, and Jiekun Yang
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0301 basic medicine ,Drug ,media_common.quotation_subject ,Science ,General Physics and Astronomy ,Mice, Nude ,Antineoplastic Agents ,Biology ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Gene Knockout Techniques ,Drug Delivery Systems ,In vivo ,Pancreatic cancer ,Cell Line, Tumor ,medicine ,CRISPR ,Animals ,Combinatorial Chemistry Techniques ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Genetic Testing ,lcsh:Science ,Genetic testing ,media_common ,Mitogen-Activated Protein Kinase Kinases ,Multidisciplinary ,medicine.diagnostic_test ,Cell Death ,Cancer ,Reproducibility of Results ,Drug Synergism ,General Chemistry ,Cell Cycle Checkpoints ,medicine.disease ,3. Good health ,Pancreatic Neoplasms ,030104 developmental biology ,Cancer cell ,Cancer research ,lcsh:Q - Abstract
Predicting the response and identifying additional targets that will improve the efficacy of chemotherapy is a major goal in cancer research. Through large-scale in vivo and in vitro CRISPR knockout screens in pancreatic ductal adenocarcinoma cells, we identified genes whose genetic deletion or pharmacologic inhibition synergistically increase the cytotoxicity of MEK signaling inhibitors. Furthermore, we show that CRISPR viability scores combined with basal gene expression levels could model global cellular responses to the drug treatment. We develop drug response evaluation by in vivo CRISPR screening (DREBIC) method and validated its efficacy using large-scale experimental data from independent experiments. Comparative analyses demonstrate that DREBIC predicts drug response in cancer cells from a wide range of tissues with high accuracy and identifies therapeutic vulnerabilities of cancer-causing mutations to MEK inhibitors in various cancer types., Predicting the response to chemotherapy is a major goal of cancer research. Here the authors use CRISPR knockout screens in pancreatic ductal adenocarcinoma cells to identify deletions synergistic with MEK inhibitors.
- Published
- 2018
21. CD47 Blockade as an Adjuvant Immunotherapy for Resectable Pancreatic Cancer
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Sara J. Adair, Alex D. Michaels, Craig L. Slingluff, Matthew G. Mullen, Timothy E. Newhook, Sarbajeet Nagdas, J. Thomas Parsons, Kodi S. Ravichandran, Timothy N. J. Bullock, Todd W. Bauer, Sho Morioka, Bernadette J. Goudreau, and Jesse B. Persily
- Subjects
0301 basic medicine ,Cancer Research ,medicine.medical_treatment ,CD47 Antigen ,Article ,Immunomodulation ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Mice, Inbred NOD ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Macrophage ,Neoplasm Metastasis ,Neoplasm Staging ,Tumor microenvironment ,biology ,business.industry ,Macrophages ,CD47 ,Cancer ,Immunotherapy ,medicine.disease ,Immunohistochemistry ,Xenograft Model Antitumor Assays ,Tumor Burden ,Pancreatic Neoplasms ,Disease Models, Animal ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Disease Progression ,Cancer research ,biology.protein ,Antibody ,business ,Adjuvant - Abstract
Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival. Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 (“don't eat me signal”) expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival. Results: In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival. Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415–25. ©2017 AACR.
- Published
- 2018
22. Apoptotic Bodies in the Pancreatic Tumor Cell Culture Media Enable Label‐Free Drug Sensitivity Assessment by Impedance Cytometry
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Sara J. Adair, John H. Moore, Carlos Honrado, Armita Salahi, Todd W. Bauer, and Nathan S. Swami
- Subjects
Cell ,Cell Culture Techniques ,Biomedical Engineering ,Article ,General Biochemistry, Genetics and Molecular Biology ,Flow cytometry ,Biomaterials ,Extracellular Vesicles ,Pancreatic tumor ,Cell Line, Tumor ,Electric Impedance ,Tumor Microenvironment ,medicine ,Humans ,Distribution (pharmacology) ,medicine.diagnostic_test ,Chemistry ,medicine.disease ,In vitro ,Microvesicles ,Pancreatic Neoplasms ,medicine.anatomical_structure ,Pharmaceutical Preparations ,Apoptosis ,Cancer research ,Cytometry - Abstract
The ability to rapidly and sensitively predict drug response and toxicity using in vitro models of patient-derived tumors is essential for assessing chemotherapy efficacy. Currently, drug sensitivity assessment for solid tumors relies on imaging adherent cells or by flow cytometry of cells lifted from drug-treated cultures after fluorescent staining for apoptotic markers. Subcellular apoptotic bodies (ABs), including microvesicles that are secreted into the culture media under drug treatment can potentially serve as markers for drug sensitivity, without the need to lift cells under culture. However, their stratification to quantify cell disassembly is challenging due to their compositional diversity, with tailored labeling strategies currently needed for the recognition and cytometry of each AB type. It is shown that the high frequency impedance phase versus size distribution of ABs determined by high-throughput single-particle impedance cytometry of supernatants in the media of gemcitabine-treated pancreatic tumor cultures exhibits phenotypic resemblance to lifted apoptotic cells and enables shape-based stratification within distinct size ranges, which is not possible by flow cytometry. It is envisioned that this tool can be applied in conjunction with the appropriate pancreatic tumor microenvironment model to assess drug sensitivity and toxicity of patient-derived tumors, without the need to lift cells from cultures.
- Published
- 2021
23. Abstract PO-007: PRMT5 inhibition sensitizes pancreatic cancer to gemcitabine in orthotopic and metastatic murine models
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Sarbajeet Nagdas, William J. Kane, Denis Liu, Sara J. Adair, Mazhar Adli, Todd W. Bauer, and Xiaolong Wei
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Cancer Research ,medicine.diagnostic_test ,business.industry ,medicine.medical_treatment ,Protein arginine methyltransferase 5 ,Splenectomy ,Cancer ,Magnetic resonance imaging ,medicine.disease ,Gemcitabine ,Oncology ,In vivo ,Pancreatic cancer ,medicine ,Cancer research ,Bioluminescence imaging ,business ,medicine.drug - Abstract
Purpose: The purpose of this study was to investigate protein arginine methyltransferase 5 (PRMT5) as a therapeutic target in combination with gemcitabine for pancreatic cancer in preclinical models. Procedures: In an orthotopic xenograft model, PRMT5 wild-type (WT) or knockout (KO) mPanc96 cells were injected into the pancreata of athymic nude mice. Mice were randomized to control or gemcitabine (100 mg/kg) arms. Tumor volume was measured with magnetic resonance imaging (MRI) at 21, 28, and 35 days post-injection. In a liver metastasis model, PRMT5 WT or KO mPanc96 cells were injected into the spleens of athymic nude mice followed by splenectomy after 10 minutes. Mice were randomized to control or gemcitabine (100 mg/kg) arms. Metastatic tumor burden within the liver was measured with in vivo bioluminescence imaging twice weekly for 19 days post-injection. Relative bioluminescence was calculated relative to post-injection day 2. Results: In the orthotopic model, untreated PRMT5 KO tumors exhibited a 46% reduction in tumor volume compared to untreated PRMT5 WT tumors (959 mm−3 vs. 1766 mm−3, p=0.009). Furthermore, gemcitabine-treated PRMT5 KO tumors exhibited a 30% reduction in tumor volume compared to gemcitabine-treated PRMT5 WT tumors (405 mm−3 vs. 579 mm−3, p=0.046). Compared to untreated PRMT5 WT tumors, gemcitabine-treated PRMT5 KO tumors exhibited a 77% reduction in tumor volume (405 mm−3 vs. 1766 mm−3, p= Citation Format: William J. Kane, Sara J. Adair, Sarbajeet Nagdas, Denis Liu, Xiaolong Wei, Mazhar Adli, Todd W. Bauer. PRMT5 inhibition sensitizes pancreatic cancer to gemcitabine in orthotopic and metastatic murine models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-007.
- Published
- 2020
24. Evaluation of SAS1B as a target for antibody-drug conjugate therapy in the treatment of pancreatic cancer
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Sara J. Adair, Todd W. Bauer, Arabinda Mandal, Kiley A. Knapp, Anne M. Mills, Craig L. Slingluff, John C. Herr, J. Thomas Parsons, Timothy N. J. Bullock, Walter C. Olson, and Eusebio S. Pires
- Subjects
0301 basic medicine ,Cellular immunity ,Antibody-drug conjugate ,Antibody-Drug Conjugate Therapy ,medicine.drug_class ,Cell ,Monoclonal antibody ,antibody-drug conjugate ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,Immunotoxin ,Pancreatic cancer ,Medicine ,business.industry ,ASTL/SAS1B/ovastacin ,surface cancer-oocyte antigen ,medicine.disease ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,pancreatic cancer biomarker ,Cancer research ,targeted immunotherapy ,business ,Research Paper - Abstract
Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 μg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.
- Published
- 2017
25. Co-Treatment with Panitumumab and Trastuzumab Augments Response to the MEK Inhibitor Trametinib in a Patient-Derived Xenograft Model of Pancreatic Cancer
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Edward B. Stelow, Todd W. Bauer, Sara J. Adair, Dustin M. Walters, Timothy E. Newhook, J. Thomas Parsons, Alison J. Kim, and James M. Lindberg
- Subjects
Trametinib ,MAPK/ERK pathway ,Cancer Research ,MEK inhibitor ,Biology ,medicine.disease_cause ,Lapatinib ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,lcsh:RC254-282 ,Epidermal growth factor ,medicine ,Cancer research ,biology.protein ,Panitumumab ,KRAS ,Epidermal growth factor receptor ,skin and connective tissue diseases ,neoplasms ,medicine.drug - Abstract
Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and epidermal growth factor receptor (EGFR) family signaling are drivers of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated that combinatorial treatment of PDAC xenografts with the mitogen-activated protein kinase–extracellular-signal-regulated kinase (ERK) kinase1/2 (MEK1/2) inhibitor trametinib and the dual EGFR/human epidermal growth factor receptor 2 (HER2) inhibitor lapatinib provided more effective inhibition than either treatment alone. In this study, we have used the therapeutic antibodies, panitumumab (specific for EGFR) and trastuzumab (specific for HER2), to probe the role of EGFR and HER2 signaling in the proliferation of patient-derived xenograft (PDX) tumors. We show that dual anti-EGFR and anti-HER2 therapy significantly augmented the growth inhibitory effects of the MEK1/2 inhibitor trametinib in three different PDX tumors. While significant growth inhibition was observed in both KRAS mutant xenograft groups receiving trametinib and dual antibody therapy (tumors 366 and 608), tumor regression was observed in the KRAS wild-type xenografts (tumor 738) treated in the same manner. Dual antibody therapy in conjunction with trametinib was equally or more effective at inhibiting tumor growth and with lower apparent toxicity than trametinib plus lapatinib. Together, these studies provide further support for a role for EGFR and HER2 in pancreatic cancer proliferation and underscore the importance of therapeutic intervention in both the KRAS–rapidly accelerated fibrosarcoma kinase (RAF)–MEK–ERK and EGFR-HER2 pathways to achieve maximal therapeutic efficacy in patients.
- Published
- 2014
26. Inhibition of the Growth of Patient-Derived Pancreatic Cancer Xenografts with the MEK Inhibitor Trametinib Is Augmented by Combined Treatment with the Epidermal Growth Factor Receptor/HER2 Inhibitor Lapatinib
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Timothy E. Newhook, Tona M. Gilmer, Bryce T. Lowrey, Todd W. Bauer, Cheryl A. Borgman, Catharine R. Cowan, Maria E. Chopivsky, Sara J. Adair, Edward B. Stelow, Jayme B. Stokes, Dustin M. Walters, James M. Lindberg, and John T. Parsons
- Subjects
MAPK/ERK pathway ,Cancer Research ,MAP Kinase Signaling System ,Pyridones ,Receptor, ErbB-2 ,AKT2 ,Antineoplastic Agents ,Pyrimidinones ,Biology ,medicine.disease_cause ,Lapatinib ,lcsh:RC254-282 ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Pancreatic cancer ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Epidermal growth factor receptor ,Phosphorylation ,Protein Kinase Inhibitors ,030304 developmental biology ,Trametinib ,0303 health sciences ,MEK inhibitor ,medicine.disease ,MAP Kinase Kinase Kinases ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,3. Good health ,ErbB Receptors ,Pancreatic Neoplasms ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Quinazolines ,KRAS ,Neoplasm Transplantation ,medicine.drug ,Signal Transduction ,Research Article - Abstract
Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.
- Published
- 2013
27. Adjuvant Trametinib Delays the Outgrowth of Occult Pancreatic Cancer in a Mouse Model of Patient-Derived Liver Metastasis
- Author
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J. Thomas Parsons, Osama E. Rahma, Alison J. Kim, James M. Lindberg, Timothy E. Newhook, Sara J. Adair, Edward B. Stelow, and Todd W. Bauer
- Subjects
0301 basic medicine ,Oncology ,medicine.medical_specialty ,endocrine system diseases ,Pyridones ,medicine.medical_treatment ,Pyrimidinones ,medicine.disease_cause ,Article ,Metastasis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Pancreatic cancer ,Internal medicine ,medicine ,Carcinoma ,Tumor Cells, Cultured ,Animals ,Humans ,Protein Kinase Inhibitors ,Trametinib ,business.industry ,MEK inhibitor ,Liver Neoplasms ,medicine.disease ,Xenograft Model Antitumor Assays ,Gemcitabine ,Pancreatic Neoplasms ,030104 developmental biology ,030220 oncology & carcinogenesis ,Surgery ,KRAS ,business ,Adjuvant ,medicine.drug ,Carcinoma, Pancreatic Ductal - Abstract
Most patients with pancreatic ductal adenocarcinoma (PDAC) die within 5 years following resection plus adjuvant gemcitabine (Gem) from outgrowth of occult metastases. We hypothesized that inhibition of the KRAS pathway with the MEK inhibitor trametinib would inhibit the outgrowth of occult liver metastases in a preclinical model. Liver metastases harvested from two patients with PDAC (Tumors 608, 366) were implanted orthotopically in mice. Tumor cell lines were derived and transduced with lentiviruses encoding luciferase and injected into spleens of mice generating microscopic liver metastases. Growth kinetics of liver metastases were measured with bioluminescent imaging and time-to-progression (TTP), progression-free survival (PFS), and overall survival (OS) were determined. Trametinib (0.3 mg/kg BID) significantly prolonged OS versus control (Tumor 608: 114 vs. 43 days, p
- Published
- 2015
28. A Thirteen-Gene Expression Signature Predicts Survival of Patients with Pancreatic Cancer and Identifies New Genes of Interest
- Author
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Todd W. Bauer, J. Thomas Parsons, Jason A. Papin, James M. Lindberg, Sara J. Adair, Jae K. Lee, Wenjun Xin, Timothy E. Newhook, and Edik M. Blais
- Subjects
Oncology ,medicine.medical_specialty ,Pancreatic ductal adenocarcinoma ,endocrine system diseases ,Genetic Causes of Cancer ,lcsh:Medicine ,Adenocarcinoma ,Bioinformatics ,Metastasis ,Pancreatic Cancer ,Breast cancer ,Pancreatic cancer ,Internal medicine ,Gene expression ,Gastrointestinal Tumors ,medicine ,Medicine and Health Sciences ,Humans ,lcsh:Science ,Gene ,Staging system ,Multidisciplinary ,business.industry ,Cancer Risk Factors ,Gene Expression Profiling ,lcsh:R ,Cancers and Neoplasms ,Reproducibility of Results ,medicine.disease ,Prognosis ,Survival Analysis ,digestive system diseases ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Pancreatic Neoplasms ,lcsh:Q ,Treatment decision making ,Lymph Nodes ,business ,Research Article ,Carcinoma, Pancreatic Ductal ,Genes, Neoplasm ,Signal Transduction - Abstract
Background Currently, prognostication for pancreatic ductal adenocarcinoma (PDAC) is based upon a coarse clinical staging system. Thus, more accurate prognostic tests are needed for PDAC patients to aid treatment decisions. Methods and Findings Affymetrix gene expression profiling was carried out on 15 human PDAC tumors and from the data we identified a 13-gene expression signature (risk score) that correlated with patient survival. The gene expression risk score was then independently validated using published gene expression data and survival data for an additional 101 patients with pancreatic cancer. Patients with high-risk scores had significantly higher risk of death compared to patients with low-risk scores (HR 2.27, p = 0.002). When the 13-gene score was combined with lymph node status the risk-score further discriminated the length of patient survival time (p
- Published
- 2014
29. Abstract A47: A patient-derived xenograft model of pancreatic cancer in mice to develop novel adjuvant therapies
- Author
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J. Thomas Parsons, Matthew G. Mullen, Edward B. Stelow, Timothy E. Newhook, Todd W. Bauer, Sara J. Adair, Alex D. Michaels, Jason A. Papin, James M. Lindberg, and Edik M. Blais
- Subjects
Trametinib ,Cancer Research ,Pathology ,medicine.medical_specialty ,business.industry ,MEK inhibitor ,Cancer ,medicine.disease ,Gemcitabine ,Metastasis ,Oncology ,Pancreatic cancer ,Cancer research ,Adjuvant therapy ,medicine ,Bioluminescence imaging ,business ,medicine.drug - Abstract
Background: Eighty percent of patients with pancreatic ductal adenocarcinoma (PDAC) die from their disease within five years of surgical resection, likely due to presence of occult metastases at diagnosis. We have developed a patient-derived xenograft (PDX) model in mice to investigate the behavior of occult metastatic cells in the liver microenvironment and derive novel adjuvant therapies. Methods: Five PDAC tumors (215, 366, 608, 654 and 738) were resected from patients and implanted orthotopically in mice. Tumors were harvested, cell lines generated and transduced with luciferase, then injected into spleens of mice to generate microscopic liver metastases, then primary tumors removed via splenectomy. Bioluminescence imaging of mice and histologic analysis and flow cytometry of livers were utilized to characterize each tumor's distinct pattern of cell clearance and outgrowth kinetics. Affymetrix gene expression of tumors was performed. Mice were treated with adjuvant therapy following resection of primary tumors in the spleen and time-to-progression (TTP) and overall survival (OS) were measured. Results: Each PDX cell line demonstrated unique and reproducible clearance in the liver and outgrowth kinetics as measured by bioluminescence imaging. Distinct differences in gene expression were identified in tumors exhibiting rapid vs. delayed outgrowth. The MEK inhibitor trametinib (0.3 mg/kg oral daily) prolonged TTP and OS vs. control (OS - Tumor 608: 114 vs. 43 days, p Conclusions: This PDX PDAC model of occult metastasis allows characterization of hepatic clearance of tumor cells and outgrowth kinetics. Metastatic outgrowth appears to be dependent upon distinguishable tumor cell-specific factors. Trametinib effectively inhibits KRAS-MEK-ERK signaling, delays outgrowth of occult metastases and prolongs survival of mice. Utilization of this model will help further define the complex interaction of PDAC cells and the metastatic microenvironment of the liver. Citation Format: Matthew G. Mullen, Timothy E. Newhook, James M. Lindberg, Sara J. Adair, Edik M. Blais, Alex D. Michaels, Edward B. Stelow, Jason A. Papin, J. Thomas Parsons, Todd W. Bauer. A patient-derived xenograft model of pancreatic cancer in mice to develop novel adjuvant therapies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A47.
- Published
- 2016
30. Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment
- Author
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J. Thomas Parsons, Keena S. Thomas, Bryce T. Lowrey, Edward B. Stelow, Sara J. Adair, Jayme B. Stokes, Jill K. Slack-Davis, E. Daniel Hershey, Dustin M. Walters, Robert W. Tilghman, Todd W. Bauer, Rosa F. Hwang, and Amy H. Bouton
- Subjects
Male ,Cancer Research ,Stromal cell ,Indoles ,Angiogenesis ,Mice, Nude ,Antineoplastic Agents ,Biology ,Article ,Metastasis ,Focal adhesion ,Mice ,Cell Movement ,Pancreatic cancer ,Cell Line, Tumor ,medicine ,Tumor Microenvironment ,Animals ,Humans ,Neoplasm Invasiveness ,Neoplasm Metastasis ,Protein Kinase Inhibitors ,Cell Proliferation ,Tumor microenvironment ,Sulfonamides ,Cell growth ,Receptor Protein-Tyrosine Kinases ,medicine.disease ,Oncology ,Focal Adhesion Protein-Tyrosine Kinases ,Cancer research ,Cancer-Associated Fibroblasts ,Carcinoma, Pancreatic Ductal ,Signal Transduction - Abstract
Current therapies for pancreatic ductal adenocarcinoma (PDA) target individual tumor cells. Focal adhesion kinase (FAK) is activated in PDA, and levels are inversely associated with survival. We investigated the effects of PF-562,271 (a small-molecule inhibitor of FAK/PYK2) on (i) in vitro migration, invasion, and proliferation; (ii) tumor proliferation, invasion, and metastasis in a murine model; and (iii) stromal cell composition in the PDA microenvironment. Migration assays were conducted to assess tumor and stromal cell migration in response to cellular factors, collagen, and the effects of PF-562,271. An orthotopic murine model was used to assess the effects of PF-562,271 on tumor growth, invasion, and metastasis. Proliferation assays measured PF-562,271 effects on in vitro growth. Immunohistochemistry was used to examine the effects of FAK inhibition on the cellular composition of the tumor microenvironment. FAK and PYK2 were activated and expressed in patient-derived PDA tumors, stromal components, and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK (phospho-FAK or Y397) in a dose-dependent manner. PF-562,271 inhibited migration of tumor cells, cancer-associated fibroblasts, and macrophages. Treatment of mice with PF-562,271 resulted in reduced tumor growth, invasion, and metastases. PF-562,271 had no effect on tumor necrosis, angiogenesis, or apoptosis, but it did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts than control or gemcitabine. These data support a role for FAK in PDA and suggest that inhibitors of FAK may contribute to efficacious treatment of patients with PDA. Mol Cancer Ther; 10(11); 2135–45. ©2011 AACR.
- Published
- 2011
31. Treatment of ovarian cancer cell lines with 5-aza-2'-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules
- Author
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Sara J. Adair and Kevin T. Hogan
- Subjects
endocrine system ,Cancer Research ,Antimetabolites, Antineoplastic ,Immunology ,Antigen presentation ,CD1 ,Gene Expression ,CD8-Positive T-Lymphocytes ,Major histocompatibility complex ,Decitabine ,Polymerase Chain Reaction ,Antigen ,Antigens, Neoplasm ,Cell Line, Tumor ,MHC class I ,medicine ,Immunology and Allergy ,Humans ,Ovarian Neoplasms ,biology ,Histocompatibility Antigens Class I ,Cancer ,DNA Methylation ,medicine.disease ,Flow Cytometry ,Up-Regulation ,Oncology ,biology.protein ,Cancer research ,Azacitidine ,Cancer/testis antigens ,Female ,CD8 - Abstract
To test the hypothesis that decrease in DNA methylation will increase the expression of cancer-testis antigens (CTA) and class I major histocompatibility complex (MHC)-encoded molecules by ovarian cancer cells, and thus increase the ability of these cells to be recognized by antigen-reactive CD8(+) T cells.Human ovarian cancer cell lines were cultured in the presence or absence of varying concentrations of the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC) for 3-7 days. The expression levels of 12 CTA genes were measured using the polymerase chain reaction. The protein expression levels of class I MHC molecules and MAGE-A1 were measured by flow cytometry. T cell reactivity was determined using interferon-gamma ELISpot analysis.DAC treatment of ovarian cancer cell lines increased the expression of 11 of 12 CTA genes tested including MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, NY-ESO-1, TAG-1, TAG-2a, TAG-2b, and TAG-2c. In contrast, DAC treatment decreased the already low expression of the MAGE-A2 gene by ovarian cancer cells, a finding not previously observed in cancers of any histological type. DAC treatment increases the expression of class I MHC molecules by the cells. These effects were time-dependent over a 7-day interval, and were dose-dependent up to 1-3 microM for CTA and up to 10 microM for class I MHC molecules. Each cell line tested had a unique pattern of gene upregulation after exposure to DAC. The enhanced expression levels increased the recognition of 2 of 3 antigens recognized by antigen-reactive CD8(+) T cells.These results demonstrate the potential utility of combining DAC therapy with vaccine therapy in an attempt to induce the expression of antigens targeted by the vaccine, but they also demonstrate that care must be taken to target inducible antigens.
- Published
- 2008
32. The TAG family of cancer/testis antigens is widely expressed in a variety of malignancies and gives rise to HLA-A2-restricted epitopes
- Author
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Sara J. Adair, Tiffany M. Carr, Mitsú J. Fink, Craig L. Slingluff, and Kevin T. Hogan
- Subjects
Cancer Research ,Lung Neoplasms ,medicine.medical_treatment ,Cell Adhesion Molecules, Neuronal ,Immunology ,Epitopes, T-Lymphocyte ,Uterine Cervical Neoplasms ,Breast Neoplasms ,Major histocompatibility complex ,Epitope ,Antigen ,Antigens, Neoplasm ,Cell Line, Tumor ,Neoplasms ,HLA-A2 Antigen ,medicine ,Contactin 2 ,Immunology and Allergy ,Cytotoxic T cell ,Humans ,Amino Acid Sequence ,Pharmacology ,Ovarian Neoplasms ,biology ,Brain Neoplasms ,Melanoma ,Histocompatibility Antigens Class I ,Cancer ,Pharyngeal Neoplasms ,Immunotherapy ,Dendritic Cells ,medicine.disease ,Cytotoxicity Tests, Immunologic ,Peptide Fragments ,Tongue Neoplasms ,Gene Expression Regulation, Neoplastic ,Colonic Neoplasms ,biology.protein ,Cancer research ,Leukocytes, Mononuclear ,Cancer/testis antigens ,Female ,T-Lymphocytes, Cytotoxic - Abstract
The TAG-1, TAG-2a, TAG-2b, and TAG-2c cancer/testis genes, known to be expressed in an unusually high percentage of melanoma cell lines, are shown here to be expressed in a variety of tumor lines of diverse histologic type, including cancers of the brain, breast, colon, lung, ovary, pharynx, and tongue. The genes are also expressed in fresh, uncultured melanoma, and ovarian cancer cells. Epitope prediction algorithms were used to identify potential HLA-A1, HLA-A2, HLA-A3, HLA-B7, and HLA-B8 epitopes, and these potential epitopes were tested for their ability to stimulate a peptide-specific cytotoxic T lymphocyte response using lymphocytes from healthy donors. Two HLA-A2-restricted epitopes (SLGWLFLLL and LLLRLECNV) were identified using this approach. Cytotoxic T lymphocytes specific for each of these peptides were capable of recognizing tumor cells expressing both the corresponding class I major histocompatibility complex encoded molecule and the TAG genes. These results indicate that TAG-derived peptides may be good components of a therapeutic vaccine designed to target melanoma and a variety of epithelial cell-derived malignancies.
- Published
- 2007
33. Immunological profiling of a panel of human ovarian cancer cell lines
- Author
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Tiffany M. Carr, Sara J. Adair, Mitsú J. Fink, and Kevin T. Hogan
- Subjects
Cancer Research ,Immunology ,Enzyme-Linked Immunosorbent Assay ,Biology ,Major histocompatibility complex ,Polymerase Chain Reaction ,Flow cytometry ,Antigen ,Antigens, Neoplasm ,Cell Line, Tumor ,medicine ,Immunology and Allergy ,Cytotoxic T cell ,Humans ,Ovarian Neoplasms ,medicine.diagnostic_test ,Histocompatibility Antigens Class I ,Histocompatibility Antigens Class II ,medicine.disease ,Flow Cytometry ,Molecular biology ,Tumor antigen ,CTL ,Oncology ,Cell culture ,biology.protein ,Cytokines ,Female ,Ovarian cancer - Abstract
The efficient identification of peptide antigens recognized by ovarian cancer-specific cytotoxic T lymphocytes (CTL) requires the use of well-characterized ovarian cancer cell lines. To develop such a panel of cell lines, 11 ovarian cancer cell lines were characterized for the expression of class I and class II major histocompatibility complex (MHC)-encoded molecules, 15 tumor antigens, and immunosuppressive cytokines [transforming growth factor beta (TGF-beta) and IL-10].Class I MHC gene expression was determined by polymerase chain reaction (PCR), and class I and class II MHC protein expression was determined by flow cytometry. Tumor antigen expression was determined by a combination of polymerase chain reaction (PCR) and flow cytometry. Cytokine expression was determined by ELISA.Each of the ovarian cancer cell lines expresses cytokeratins, although each cell line does not express the same cytokeratins. One of the lines expresses CD90, which is associated with a fibroblast lineage. Each of the cell lines expresses low to moderate amounts of class I MHC molecules, and several of them express low to moderate amounts of class II MHC molecules. Using a combination of PCR and flow cytometry, it was determined that each cell line expressed between six and thirteen of fifteen antigens tested. Little to no TGF-beta3 was produced by any of the cell lines, TGF-beta1 was produced by three of the cell lines, TGF-beta2 was produced by all of the cell lines, with four of the cell lines producing large amounts of the latent form of the molecule, and IL-10 was produced by one of the cell lines.Each of the 11 ovarian cancer lines is characterized by a unique expression pattern of epithelial/fibroblast markers, MHC molecules, tumor antigens, and immunosuppressive cytokines. Knowledge of these unique expression patterns will increase the usefulness of these cell lines in identifying the antigens recognized by ovarian cancer-specific CTL.
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- 2007
34. Abstract B21: In a patient-derived xenograft model of occult hepatic metastasis from pancreatic cancer the MEK inhibitor trametinib delays tumor outgrowth and prolongs survival
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James M. Lindberg, Timothy E. Newhook, Sara J. Adair, Todd W. Bauer, and J. Thomas Parsons
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Trametinib ,Cancer Research ,ved/biology ,business.industry ,medicine.medical_treatment ,MEK inhibitor ,ved/biology.organism_classification_rank.species ,Splenectomy ,medicine.disease ,Oncology ,Apoptosis ,Tumor progression ,Pancreatic cancer ,Adjuvant therapy ,medicine ,Cancer research ,Model organism ,business ,Molecular Biology - Abstract
Background: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and the majority of patients succumb to metastatic disease. Even for those with localized PDAC, most will die from metastatic disease despite margin-negative resection and adjuvant therapy. Therefore, these patients must harbor occult metastatic PDAC at presentation. There is a compelling need for the development of preclinical models that efficiently recapitulate occult metastatic liver PDAC to identify molecular and cellular pathways that drive metastatic cellular survival and growth. Towards this aim, we have developed a PDAC model of occult liver metastases using patient-derived xenografts (PDXs) to study the growth of PDAC within the metastatic environment and evaluated the effect of MEK inhibitor therapy on tumor progression. Methods and Results: Extensively characterized patient-derived KRAS-mutant (Tumors 608, 366, and 654) and wild-type (Tumors 738 and 215) PDAC cells were transduced with luciferase and injected into the spleens of athymic, nude mice, allowed to circulate for 10 minutes, after which a splenectomy was performed. To evaluate metastatic cell growth kinetics in the liver, tumor burden was monitored by sequential bioluminescent imaging. All mice exhibited defined phases of survival/dormancy and the proliferative outgrowth; however, differential kinetics were observed for each tumor cell line. To evaluate the effect of MEK inhibition of occult metastatic PDAC cells in the liver, Tumor 608 cells were injected and mice either received the MEK inhibitor trametinib (0.3 mg/kg, daily) or vehicle control beginning 48 hour post-injection. Trametinib significantly reduced metastatic tumor burden, delayed time to proliferative outgrowth, and greatly prolonged survival as compared to control (med. survival: 114 vs. 43 days, p Conclusions: In a model of occult liver metastatic PDAC, patient-derived tumors exhibit different growth kinetics in the liver environment. Furthermore, MEK inhibition with trametinib decreased metastatic cellular proliferation, increased apoptosis, prolonged metastatic tumor outgrowth, and significantly increased survival of mice harboring occult hepatic metastases from PDAC. This efficacy of single agent trametinib is unique to occult metastatic disease and is not seen in orthotopic models of advanced, established pancreatic tumors. This finding illustrates the importance of using specific preclinical models that best recapitulate the clinical aspects of patients who will be evaluated in future clinical trials. Further investigation into the cellular and molecular factors promoting PDAC cell survival within the hepatic microenvironment utilizing this model will lead to development of rational therapeutic strategies for patients with occult metastatic disease. Citation Format: Timothy Newhook, James Lindberg, Sara Adair, J. Thomas Parsons, Todd W. Bauer. In a patient-derived xenograft model of occult hepatic metastasis from pancreatic cancer the MEK inhibitor trametinib delays tumor outgrowth and prolongs survival. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B21.
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- 2014
35. Abstract 808: Combination therapy with a MEK inhibitor plus T-type calcium channel inhibitor is highly effective in patient-derived pancreatic ductal adenocarcinomas
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J. Thomas Parsons, Timothy E. Newhook, Jason A. Papin, James M. Lindberg, Sara J. Adair, Lloyd S. Gray, Edik M. Blais, and Todd W. Bauer
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Trametinib ,Cancer Research ,Mibefradil ,endocrine system diseases ,Combination therapy ,business.industry ,MEK inhibitor ,T-type calcium channel ,Cancer ,Pharmacology ,medicine.disease ,medicine.disease_cause ,digestive system diseases ,chemistry.chemical_compound ,Oncology ,chemistry ,medicine ,Cancer research ,KRAS ,Growth inhibition ,business ,medicine.drug - Abstract
Introduction: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and novel therapeutic strategies are needed. Mutations in the KRAS oncogene are important drivers of PDAC progression; however, monotherapies targeting the RAS pathway have shown only modest efficacy. The RAS pathway is an activator of calcium (Ca2+) signaling, which has been implicated in PDAC progression. We hypothesized that MEK inhibition combined with the T-type calcium channel inhibitor mibefradil would result in enhanced growth inhibition of PDAC tumors. Methods and Results: Gene expression profiling of patient-derived PDAC tumors (relative to normal pancreas) and tumors chronically treated with the MEK inhibitor trametinib in vivo revealed an upregulation of Ca2+-signaling related genes including members of the calpain pathway and the calmodulin pathway, both implicated in PDAC progression. To assess the in vivo efficacy of combined T-type calcium channel inhibition and MEK inhibition, mice were engrafted orthotopically with patient-derived KRAS-mutant (Tumor 366) and KRAS-wild type (Tumor 738) PDAC tumors and treated with control, the MEK inhibitor trametinib, the T-type calcium channel inhibitor mibefradil, or combination. Combination therapy with trametinib plus mibefradil was highly effective with greater inhibition of PDAC growth than either therapy alone in the KRAS-mutant Tumor 366, however mibefradil did not augment the level of growth inhibition achieved by trametinib alone in the KRAS-wild type Tumor 738. To evaluate combination trametinib plus mibefradil therapy in patient-derived PDAC tumors following chronic treatment with trametinib, mice were engrafted orthotopically with patient-derived PDAC tumors, allowed to grow to 300-500mm3, and treated with trametinib for 4-6 weeks. Following initial treatment, tumors were harvested, re-implanted, and again exposed to therapy over 4-6 weeks. The cycles of treatment and reimplantation were continued until tumor growth was not significantly reduced by trametinib treatment. Therapy with trametinib plus mibefradil nearly completely inhibited growth in KRAS-mutant PDAC tumors (Tumors 608 and 366) previously resistant to chronic trametinib therapy. Conclusions: Combination therapy with the MEK inhibitor trametinib plus the T-type calcium channel inhibitor mibefradil results in significant growth inhibition of KRAS-mutant patient-derived PDAC tumors. Moreover, this combination therapy results in near complete growth inhibition of PDAC tumors that have acquired resistance to trametinib. The combination therapy of mibefradil plus trametinib should be further evaluated in clinical trials for patients with PDAC. Citation Format: Timothy Eric Newhook, James M. Lindberg, Sara J. Adair, Edik Blais, Jason Papin, Lloyd Gray, J. Thomas Parsons, Todd W. Bauer. Combination therapy with a MEK inhibitor plus T-type calcium channel inhibitor is highly effective in patient-derived pancreatic ductal adenocarcinomas. [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 808. doi:10.1158/1538-7445.AM2014-808
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- 2014
36. Abstract 4040: The MEK inhibitor trametinib delays tumor outgrowth and prolongs survival in a patient-derived mouse model of occult hepatic metastatic pancreatic cancer
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Timothy E. Newhook, James M. Lindberg, Todd W. Bauer, J. Thomas Parsons, and Sara J. Adair
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Trametinib ,Cancer Research ,Pathology ,medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,MEK inhibitor ,Splenectomy ,Cancer ,medicine.disease_cause ,medicine.disease ,Oncology ,Tumor progression ,Apoptosis ,medicine ,Cancer research ,Adjuvant therapy ,KRAS ,business - Abstract
Background: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and the majority of patients succumb to metastatic disease. Even for those with localized PDAC, most will die from metastatic disease despite margin-negative resection and adjuvant therapy. Therefore, these patients must harbor occult metastatic PDAC at presentation. We have developed a PDAC model of occult liver metastases using patient-derived xenografts (PDXs) to study the growth of PDAC within the metastatic microenvironment of the liver and evaluated the role of KRAS-MEK-ERK signaling on tumor progression. Methods and Results: Extensively characterized low passage, patient-derived KRAS-mutant (Tumors 608, 366, and 654) and wild-type (Tumors 738 and 215) PDAC cells expressing luciferase were injected into the spleens of athymic, nude mice and allowed to circulate for 10 minutes, after which a splenectomy was performed. To evaluate metastatic cell growth kinetics in the liver, tumor burden was monitored by sequential bioluminescent imaging. Each of the PDX tumors exhibited a characteristic and reproducible time to proliferative outgrowth ranging from 20 days (Tumor 608) to greater than 100 days (Tumor 654). To evaluate the role of KRAS signaling in maintaining dormant cell survival and proliferative outgrowth, tumor 608 cells were injected and mice were treated with the MEK inhibitor trametinib (0.3 mg/kg, daily) or control beginning 48 hours post-injection. Trametinib significantly reduced metastatic tumor burden, delayed time to proliferative outgrowth, and greatly prolonged survival as compared to control (med. survival: 114 vs. 43 days, p Conclusions: Using a model of occult liver metastatic PDAC, patient-derived tumors exhibited characteristic, albeit different growth kinetics in the liver microenvironment. Further, inhibition of KRAS-MEK-ERK signaling with the MEK inhibitor trametinib decreased metastatic cellular proliferation, increased apoptosis, prolonged metastatic tumor outgrowth, and significantly increased survival. Further investigation into factors promoting PDAC cell survival within the hepatic microenvironment will lead to development of rational therapeutic strategies for patients with occult metastatic PDAC. Citation Format: Timothy Eric Newhook, James M. Lindberg, Sara J. Adair, J. Thomas Parsons, Todd W. Bauer. The MEK inhibitor trametinib delays tumor outgrowth and prolongs survival in a patient-derived mouse model of occult hepatic metastatic pancreatic cancer. [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 4040. doi:10.1158/1538-7445.AM2014-4040
- Published
- 2014
37. Clinical, Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens
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Sara J. Adair, Jayme B. Stokes, J. Thomas Parsons, Jae K. Lee, Bryce T. Lowrey, Cheryl A. Borgman, Todd W. Bauer, Edik M. Blais, Dustin M. Walters, Wenjun Xin, Edward B. Stelow, and Jason A. Papin
- Subjects
Pathology ,endocrine system diseases ,lcsh:Medicine ,Kaplan-Meier Estimate ,Mice, SCID ,Disease ,Mice ,0302 clinical medicine ,Mice, Inbred NOD ,Cluster Analysis ,lcsh:Science ,Peritoneal Neoplasms ,Oligonucleotide Array Sequence Analysis ,Smad4 Protein ,0303 health sciences ,Multidisciplinary ,Liver Neoplasms ,3. Good health ,ErbB Receptors ,Gene Expression Regulation, Neoplastic ,030220 oncology & carcinogenesis ,Cytokines ,Adenocarcinoma ,Carcinoma, Pancreatic Ductal ,Research Article ,medicine.medical_specialty ,Pancreatic ductal adenocarcinoma ,Transplantation, Heterologous ,Biology ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Humans ,030304 developmental biology ,Gene Expression Profiling ,lcsh:R ,Disease progression ,Key features ,medicine.disease ,digestive system diseases ,Pancreatic Neoplasms ,Gene expression profiling ,Mutation ,ras Proteins ,Cancer research ,lcsh:Q ,Tumor Suppressor Protein p53 ,Neoplasm Transplantation - Abstract
Background Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC. Methods Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression. Results Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines. Conclusions The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.
- Published
- 2013
38. Targeting occult metastatic disease: A hematogenously derived xenograft model of human pancreatic tumor growth in the murine liver
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Timothy E. Newhook, Robert W. Tilghman, Sara J. Adair, J. Thomas Parsons, James M. Lindberg, and Todd W. Bauer
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Trametinib ,Cancer Research ,Pathology ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,medicine.disease ,Gemcitabine ,Flow cytometry ,Metastasis ,Oncology ,Pancreatic tumor ,Cell culture ,Pancreatic cancer ,medicine ,Bioluminescence imaging ,business ,medicine.drug - Abstract
198 Background: Most pancreatic cancer patients will die following surgery due to recurrent metastatic disease. Thus, better systemic therapies are needed to treat occult metastases to improve survival. We have developed a model of occult liver metastasis from pancreatic cancer in order to evaluate novel treatment strategies. Methods: Pancreatic cancer cells (MAD 09-366, 08-608, MPanc96) transduced with green fluorescent protein (GFP) and luciferase were injected into the spleens of athymic, nude mice to generate hepatic metastases. Ninety-six hours after injection, tumor-bearing mice were treated with MEK1/2 inhibitor (trametinib, 0.3mg/kg, daily), gemcitabine (100mg/kg, twice weekly), or vehicle control. Sequential bioluminescence imaging, flow cytometry, and histologic evaluation were used to assess hepatic tumor growth and behavior. Results: All injected cell lines generated hepatic metastases. Different cell lines exhibited different growth kinetics. MPanc96 injected mice demonstrated a 64% decrease in average luminescence from 6 to 72 hrs after injection followed by a 146-fold increase from 72hrs until sacrifice at 21 days. Extensive liver metastases were noted at necropsy. Flow cytometry analysis of digested mouse livers following MPanc96 injection revealed rapid clearance followed by sharp outgrowth of tumor cells that mirror the bioluminescent imaging data. MPanc96 injected mice treated with trametinib exhibited significant hepatic tumor growth inhibition relative to gemcitabine and control treated mice. At 21 days, trametinib treated mice demonstrated a 14.9-fold increase in average luminescence while on treatment compared to an 82.5-fold increased for gemcitabine treated mice (p = 0.028) and a 195.5-fold increase for control mice (p = 0.027). Conclusions: This in vivo model of pancreatic liver metastases has proven effective in assessing treatment effects on pancreatic metastatic growth. Trametinib appears to be a superior agent to gemcitabine at inhibiting metastatic pancreatic tumor growth and its effectiveness will be evaluated using this model with additional patient-derived tumor cell lines.
- Published
- 2013
39. Effect of trametinib in combination with panitumumab and trastuzumab on tumor growth in an orthotopic xenograft model of human pancreatic cancer
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James M. Lindberg, Timothy E. Newhook, Sara J. Adair, Alison J. Kim, J. Thomas Parsons, and Todd W. Bauer
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MAPK/ERK pathway ,Trametinib ,Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,medicine.disease ,medicine.disease_cause ,chemistry.chemical_compound ,chemistry ,Trastuzumab ,Internal medicine ,Pancreatic cancer ,medicine ,Panitumumab ,KRAS ,Growth inhibition ,business ,medicine.drug ,EGFR inhibitors - Abstract
190 Background: Aberrant MAPK and EGFR family signaling are key drivers of pancreatic ductal adenocarcinoma(PDAC). We hypothesized that combination trametinib(MEK1/2 inhibitor), panitumumab(EGFR inhibitor) and trastuzumab(Her2 inhibitor) would more effectively suppress tumor growth than any of these monotherapies. Methods: Patient-derived PDAC cell line MAD09-366 was exposed to trametinib, panitumumab, trastuzumab, and combination therapies in vitro. Western blot analysis was performed on treated cell lysates. Athymic, nude mice were orthotopically implanted with patient-derived PDAC xenografts(MAD09-366, 08-608, and 08-738). Established murine tumors were treated with control, trametinib (0.3mg/kg, qDay), panitumumab (500ug, BIW), trastuzumab (200ug, BIW) or in combination. MRI was used to assess tumor response. Results: Two of 3 PDACs were Kras mutant, 2 of 3 demonstrated increased Her2 activity, and all 3 showed increased EGFR activity. In vitro studies showed increased growth inhibition of triple-therapy-treated cells relative to control or each inhibitor alone. Western blot analysis revealed that EGF stimulation increased Ras pathway signaling in this Kras-mutant cell line. With EGF stimulation, the greatest Ras pathway signaling inhibition was seen in triple-therapy-treated cells. In vivo studies in all PDAC xenografts revealed that triple therapy significantly decreased tumor growth rate relative to control, trametinib alone, panitumumab alone, or panitumumab plus trastuzumab. In 2 of 3 PDACs assessed, triple therapy was superior to trametinib plus panitumumab. Average tumor size in MAD08-738 triple-therapy-treated mice decreased by 9.3%. Conclusions: Triple therapy with trametinib, panitumumab, and trastuzumab demonstrated the greatest in vitro Ras signaling blockade. In vivo, this combination produced significant tumor growth inhibition or regression in all PDAC tumors studied. This regimen should be considered for a future clinical trial in pancreatic cancer patients.
- Published
- 2013
40. Abstract 5600: Acquired resistance of pancreatic cancers to combination therapy with lapatinib plus the MEK 1/2 inhibitor GSK1120212: Using a murine orthotopic xenograft model to identify resistance pathways
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James M. Lindberg, Jae K. Lee, Blais Edik, Wenjun Xin, J. Thomas Parsons, Jayme B. Stokes, Dustin M. Walters, Hanna K. Sanoff, Cheryl A. Borgman, Sara J. Adair, Todd W. Bauer, Edward B. Stelow, and Jason A. Papin
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Cancer Research ,Pathology ,medicine.medical_specialty ,Combination therapy ,business.industry ,Cancer ,Drug resistance ,Lapatinib ,medicine.disease ,medicine.disease_cause ,Gene expression profiling ,Oncology ,Epidermal growth factor ,Pancreatic cancer ,medicine ,Cancer research ,KRAS ,business ,medicine.drug - Abstract
Background: The high incidence of KRAS mutations in human pancreatic cancers has driven the testing of MEK1/2 inhibitors (e.g. GSK1120212) in clinical trials for pancreatic cancer. We have recently shown that human patient-derived tumors expressing activated epidermal growth factor (EGF)/HER2 receptors exhibit enhanced response to combination therapy with GSK1120212 and lapatinib, using a mouse xenograft model. A phase I clinical trial of GSK1120212 plus lapatinib is now underway. We report here the analysis of tumors that have acquired resistance to this therapy in the setting of the mouse xenograft model with the goal of identifying additional effective combination therapies and improved strategies for treatment of patients with resistant disease. Methods: Three patient-derived pancreatic cancer xenografts expressing either mutant or wild type KRAS and activated EGF/HER2 receptors were implanted orthotopically in nude mice and treated with combination lapatinib (EGFR/HER2 inhibitor) and GSK1120212 (MEK1/2 inhibitor). Tumor volume was assessed by sequential MRI. Following treatment, tumors were reimplanted in second and third generation mice and retreated to generate therapy-resistant tumors. Drug-resistant and drug-naïve tumors were compared using gene expression profiling, protein kinase arrays and Western blotting analysis. Results: Acquired resistance developed in all three tumor xenografts. Gene expression profiling identified multiple genes whose expression changed in response to drug treatment. Western blot analysis of tumor lysates from resistant and naive tumors revealed examples of select resistant tumors with either sustained activation of ERK1/2 or inactivation of ERK1/2 in the presence of inhibitor. Individual resistant tumors exhibiting inactivation of ERK1/2 exhibited increased activity of alternate survival signaling pathways including increased pAkt, pp38, pJNK, and pGSK3β levels relative to drug naïve control tumors. Phospho-RTK array analysis revealed increased pFGFR1 and pVEGFR1 levels in select resistant tumors suggesting a possible role for up-regulated RTK signaling in acquired resistance. Conclusions: Using an orthotopic murine model bearing patient-derived pancreatic cancer xenografts, we have developed a model of acquired resistance to lapatinib plus GSK1120212. Interrogation of comparative gene expression and phospho-protein studies revealed alterations in signaling pathways that may contribute to drug resistance. Current studies aim to exploit these pathways to 1) derive strategies for treatment of drug-resistant tumors, and 2) develop additional rational combinatorial therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5600. doi:1538-7445.AM2012-5600
- Published
- 2012
41. Acquired resistance to combination therapy with lapatinib and MEK 1/2 inhibitor GSK1120212 in an in vivo murine model of pancreatic cancer
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J. Thomas Parsons, Maria E. Chopivsky, Edward B. Stelow, Catharine R. Cowan, James M. Lindberg, Cheryl A. Borgman, Dustin M. Walters, Bryce T. Lowrey, Jayme B. Stokes, Todd W. Bauer, and Sara J. Adair
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Cancer Research ,Pathology ,medicine.medical_specialty ,biology ,Combination therapy ,Oncogene ,business.industry ,Lapatinib ,medicine.disease ,medicine.disease_cause ,Receptor tyrosine kinase ,Oncology ,Pancreatic tumor ,Pancreatic cancer ,biology.protein ,medicine ,Cancer research ,KRAS ,business ,Tyrosine kinase ,medicine.drug - Abstract
208 Background: Mutations of the oncogene KRAS and activation of cell-surface receptor tyrosine kinases are important and preserved mechanisms of tumorgenicity in pancreatic cancer. Dual inhibition of the downstream KRAS effector MEK 1/2 and tyrosine kinases EGFR and Her2 results in effective inhibition of patient-derived tumor growth in a murine orthotopic transplantation model. Because combinatorial therapies are moving rapidly into clinical trials, we sought to develop a model of acquired tumor resistance to this combination therapy. Methods: Patient-derived pancreatic tumor xenografts MAD 09-366 (KRAS mut), MAD 08-608 (KRAS mut) and MAD 08-738 (KRAS wt) were implanted orthotopically in nude mice and treated with combination lapatinib (EGFR/Her2 inhibitor) and GSK1120212 (MEK1/2 inhibitor) and tumor volume was measured by MRI. Following 4-6wks of treatment, tumors were reimplanted in second and third generation mice and retreated. Tumors were evaluated by phospho-RTK and phospho-MAPKinase array. Results: Acquired resistance developed in all three tumor xenografts. Treated tumors demonstrated a relative volume decrease in the original (F0) generation. All second re-implantation (F2) tumors, demonstrated relative tumor volume increases despite treatment. A comparison of pre-treatment mean tumor volumes showed a significant decrease in tumor size from the F0 to F2 generations suggesting selection for slower growing tumors. Array data demonstrated increased activation of FGFR1, VEGFR1/3, GSK-3β, p38, and Akt in resistant tumors as compared to their pre-treatment controls. These may represent mechanisms of tumor resistance and warrant further investigation. Conclusions: Repeated tumor exposure in vivo to combination treatment with GSK1120212 and lapatinib was used to develop a preclinical, orthotopic murine model of acquired drug resistance in patient-derived pancreatic cancers. This model provides the opportunity to define the mechanism of resistance in an appropriate tumor microenvironment and to develop alternative strategies for treating tumors resistant to this and other emerging therapies.
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- 2012
42. Abstract 1592: Targeting proliferative signals in patient-derived pancreatic cancers propagated orthotopically in immunocompromised mice
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Cheryl A. Borgman, Catharine R. Cowan, Jayme B. Stokes, Todd W. Bauer, J. Thomas Parsons, Bryce T. Lowrey, Sara J. Adair, Dustin M. Walters, Tona M. Gilmer, and Edward B. Stelow
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Cancer Research ,business.industry ,MEK inhibitor ,Cancer ,medicine.disease ,Lapatinib ,medicine.disease_cause ,chemistry.chemical_compound ,Oncology ,chemistry ,Pancreatic cancer ,Cancer research ,Medicine ,KRAS ,Erlotinib ,Growth inhibition ,business ,EGFR inhibitors ,medicine.drug - Abstract
Pancreatic ductal adenocarcinoma (PDAC) is associated with the shortest survival duration of any solid cancer. To improve treatment for this disease, better understanding of the proliferative signals that drive pancreatic cancer progression is imperative. To this end, we have established a transplantation model in which surgically resected human pancreatic cancers are propagated orthotopically in the pancreas of immunocompromised mice, permitting detailed molecular and cellular analysis. Approximately 70-85% of engrafted tumors grow and can be serially propagated in multiple generations of mice. Tumor architecture, desmoplastic character, and the metastatic properties of tumors are preserved between the original patient tumor and orthopically propagated tumors. The initial cohort of tumors reported herein was comprised of 4 early stage (Ib/IIb) and 3 late stage (IV) tumors. Five of 7 tumors had KRAS mutations in codon 12. Interrogation of tumor lysates with phospho-receptor tyrosine kinase arrays (R & D Systems) revealed activation of EGFR in all tumors, ERB2/HER2 receptors in 2 of 7 tumors, FGFR1/3 in 6 of 7 tumors, and Tie-2 receptor in 4 of 7 tumors. Based on the ubiquitous expression of EGFR family receptors and the presence of activated KRAS we assessed the efficacy of EGFR and MEK inhibitors on pancreatic cancer cell growth in culture and in the orthotopic xenograft model. A subset of patient-derived tumors adapted for growth in cell culture as well as 4 well established PDAC cell lines (MPanc96, L3.6pl, BxPC-3, PANC-1) were treated with erlotinib (EGFR inhibitor) or lapatinib (EGFR/HER2 inhibitor) and cell proliferation was assessed in a 5 day growth assay. None of the pancreatic cancer cell lines exhibited significant growth inhibition to either drug at concentrations known to be inhibitory in a sensitive cell line, SKBr3. In contrast, treatment of the pancreatic cancer cells with lapatinib in addition to a MEK inhibitor, GSK1120212, significantly inhibited cell growth in all but one of the cell lines tested. Three patient-derived tumors, including both KRAS wild type and mutant tumors, were tested in the orthotopic xenograft model for sensitivity to lapatinib, GSK1120212, or the combination of the two inhibitors. Treatment with lapatinib induced modest inhibition of tumor growth (approximately 30-50%), GSK1120212 treatment led to an approximate 60-80% reduction in tumor volume, whereas treatment with both lapatinib and GSK1120212 led to tumor regression in two tumors and greater than 80% inhibition in another. These data indicate that simultaneous inhibition of the EGF receptor and MAP kinase pathways may prove efficacious in inhibiting tumor proliferation in some primary PDACs. The effectiveness of this approach in inhibiting growth of other patient-derived tumors is under study. Supported by funding from NCI, ACS and UVa Cancer Center. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1592. doi:10.1158/1538-7445.AM2011-1592
- Published
- 2011
43. Abstract LB-241: Cancer-associated fibroblasts modulate growth- and metastasis-promoting pathways in human pancreatic cancer cells and augment orthotopic tumor growth in mice
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Bryce T. Lowrey, Todd W. Bauer, Sara J. Adair, Dustin M. Walters, Jayme B. Stokes, and J. Thomas Parsons
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Cancer Research ,Pathology ,medicine.medical_specialty ,Angiogenesis ,Cell growth ,business.industry ,medicine.medical_treatment ,Cancer ,medicine.disease ,Metastasis ,Cytokine ,Oncology ,Tumor progression ,Pancreatic cancer ,Cancer cell ,Cancer research ,medicine ,business - Abstract
Background: The role of cancer-associated fibroblasts (CAFs) in the development, progression, and metastasis of pancreatic ductal adenocarcinoma (PDAC) is not completely understood. We sought to better characterize the role of CAFs by evaluating the effect of co-culturing CAFs and pancreatic cancer cells on growth and metastasis pathways. Methods: Resected human PDACs were grown orthotopically in mice. From these tumors, human pancreatic cancer cells and mouse CAFs were isolated and grown in culture. Three cell culture environments were created: 1) pure (>99%) human pancreatic cancer cells, 2) pure (97%) mouse CAFs, and 3) co-culture of both cell types. IHC and FACS were used to confirm cell type and species of origin. Phospho-receptor tyrosine kinase (pRTK) and cytokine arrays were performed on the three groups. Additionally, the effect of CAFs on PDAC growth was evaluated in nude mice by orthotopically injecting cancer cells (1 × 106 cells) with and without CAFs (1 × 106 cells). Results: CAFs stained positively for -smooth muscle actin (-SMA) but were negative for E-cadherin, while cancer cells stained positively for E-cadherin, but not -SMA. FACS for MHC-I confirmed mouse origin of the CAFs (negative) and human origin of the cancer cells (positive). Co-culture of mouse fibroblasts and human tumor cells led to significant upregulation of cell proliferation pathways (evidenced by increased human pRTKs and cytokines, including EGFR1, EGFR2, c-Met, Ron, IGF-1R, and thrombospondin-2), angiogenic pathways (evidenced by increased secretion of VEGF, IL-8, and angiogenin) and matrix degradation and remodeling pathways (increased secretion of MMPs 8 and 9, TIMPs 1 and 4, and uPA). Orthotopic mouse injection of human pancreatic cancer cells in combination with mouse CAFs (1:1 ratio) led to larger tumors at 3 weeks as compared to injection with cancer cells alone (1129 mm3 vs. 443 mm3, p < 0.005). Conclusions: Using co-culture of CAFs and pancreatic cancer cells we have recapitulated the modulation of pathways responsible for cell proliferation, angiogenesis, and matrix remodeling thought to occur in vivo during tumor progression. Further, we show that co-injection of CAFs and pancreatic cancer cells significantly augments orthotopic tumor growth in mice. These studies lay the groundwork for future studies to define the critical factors produced by stromal fibroblasts and the role of such factors in PDAC progression, allowing new insights into improving patient therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-241.
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- 2010
44. Abstract LB-158: A human xenograft model for developing personalized targeted therapy for pancreatic cancer
- Author
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J. Thomas Parsons, Sara J. Adair, Jay W. Fox, Cheryl A. Borgman, Jason A. Papin, Dustin M. Walters, Jayme B. Stokes, Reid B. Adams, Bryce T. Lowrey, and Todd W. Bauer
- Subjects
Cancer Research ,Tumor microenvironment ,business.industry ,medicine.medical_treatment ,Cancer ,medicine.disease ,medicine.disease_cause ,Bioinformatics ,Targeted therapy ,Gene expression profiling ,medicine.anatomical_structure ,Oncology ,Pancreatic cancer ,Cancer research ,medicine ,KRAS ,Pancreas ,business ,Tyrosine kinase - Abstract
Background: Effective treatment regimens for pancreatic ductal adenocarcinoma (PDAC) are currently lacking. Molecular-targeted therapies, while promising, have been ineffective likely due to the heterogeneity and redundancy of molecular signaling pathways. Identification of predictors of response to defined targeted therapies is critical for the development of effective and personalized therapies. Methods: We have established a human PDAC tumor bank and have orthotopically implanted patient tumors into the pancreas of immunocompromised mice. Clinicopathologic data has been collected for each patient's tumor. Tumors successfully propagated in mice are routinely screened for six genetic mutations commonly found in pancreatic cancer. Gene expression profiling and phospho-receptor tyrosine kinase (pRTK) arrays, cytokine arrays, and reverse phase protein arrays (RPPAs) are used to profile the activated signaling pathways in individual tumors. A unique genetic and molecular profile is then constructed for each human tumor. Ingenuity pathway analysis software (Ingenuity Systems, Inc., Redwood, CA) is being implemented to map the unique signaling network for each tumor using the genetic and molecular data. Results: To date 7 tumors have been analyzed, including 2 metastatic tumors and 5 primary adenocarcinomas. All tumors contained TP53 gene mutation, whereas 5 tumors exhibited mutated KRAS and 3 contained mutations in the SMAD4 gene. Analysis using pRTK arrays revealed universal activation of EGFR and variable activation of ErbB2/Her2, RON, Tie-2, FGF-R1 and 3, and Axl. The profile of pRTKs appeared stable upon tumor passage both in vivo and in culture. RPPAs revealed significant variation among tumors in activation for MAPK, AKT, SAPK, and p38. Additional cytokine/growth factor array analysis and gene expression profiling is currently underway. Conclusion: The establishment of an orthotopic human pancreatic cancer xenograft model allows for the systematic analysis of genetic and molecular characteristics of human tumors propagated within a relevant tumor microenvironment. This model affords the ability to assess tumor growth responses to targeted therapies currently in use for multiple malignancies and to correlate tumor response with genetic perturbations, changes in gene expression and activation of cell signaling networks. This model will be a significant step toward developing personalized targeted therapy for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-158.
- Published
- 2010
45. Identification of Cytotoxic T Lymphocyte Epitopes Derived From the Cancer/Testis Antigen, TAG
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Sara J. Adair, Kevin T. Hogan, and Tiffany M. Carr
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Pharmacology ,Cancer Research ,Immunology ,Cancer research ,Immunology and Allergy ,Cytotoxic T cell ,Cancer/testis antigens ,Identification (biology) ,Biology ,Epitope - Published
- 2005
46. A thirteen-gene expression signature predicts survival of patients with pancreatic cancer and identifies new genes of interest.
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Timothy E Newhook, Edik M Blais, James M Lindberg, Sara J Adair, Wenjun Xin, Jae K Lee, Jason A Papin, J Thomas Parsons, and Todd W Bauer
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Medicine ,Science - Abstract
Currently, prognostication for pancreatic ductal adenocarcinoma (PDAC) is based upon a coarse clinical staging system. Thus, more accurate prognostic tests are needed for PDAC patients to aid treatment decisions.Affymetrix gene expression profiling was carried out on 15 human PDAC tumors and from the data we identified a 13-gene expression signature (risk score) that correlated with patient survival. The gene expression risk score was then independently validated using published gene expression data and survival data for an additional 101 patients with pancreatic cancer. Patients with high-risk scores had significantly higher risk of death compared to patients with low-risk scores (HR 2.27, p = 0.002). When the 13-gene score was combined with lymph node status the risk-score further discriminated the length of patient survival time (p
- Published
- 2014
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- View/download PDF
47. Clinical, molecular and genetic validation of a murine orthotopic xenograft model of pancreatic adenocarcinoma using fresh human specimens.
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Dustin M Walters, Jayme B Stokes, Sara J Adair, Edward B Stelow, Cheryl A Borgman, Bryce T Lowrey, Wenjun Xin, Edik M Blais, Jae K Lee, Jason A Papin, J Thomas Parsons, and Todd W Bauer
- Subjects
Medicine ,Science - Abstract
BACKGROUND:Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC. METHODS:Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression. RESULTS:Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines. CONCLUSIONS:The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.
- Published
- 2013
- Full Text
- View/download PDF
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