Publications by authors named "Haiyong Han"

71 Publications

Analysis of the Role of Plasma 25-Hydroxyvitamin D Levels in Survival Outcomes in Patients from the Phase III MPACT Trial of Metastatic Pancreatic Cancer.

Oncologist 2021 04 11;26(4):e704-e709. Epub 2021 Jan 11.

Bristol Myers Squibb Company, Princeton, New Jersey, USA.

Background: We examined overall survival (OS) outcomes based on plasma 25-hydroxyvitamin D [25(OH)D] levels in this post hoc analysis of the phase III MPACT trial of metastatic pancreatic cancer.

Materials And Methods: Patients were subdivided based on 25(OH)D level: sufficient (≥30 ng/mL), relatively insufficient (20-<30 ng/mL), or insufficient (<20 ng/mL).

Results: Of 861 patients randomized in MPACT, 422 were included in this analysis. In the all-patients group, the median OS among those with insufficient, relatively insufficient, and sufficient 25(OH)D levels was 7.9, 9.4, and 7.8 months, respectively. No statistically significant OS difference was observed with relatively insufficient (p = .227) or sufficient (p = .740) versus insufficient 25(OH)D levels or with sufficient vs relatively insufficient (p = .301) 25(OH)D levels.

Conclusion: No association was observed between plasma 25(OH)D levels and survival. Further investigations are needed to understand any role of vitamin D in pancreatic cancer. Clinical trial identification number. NCT00844649.
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http://dx.doi.org/10.1002/onco.13645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018329PMC
April 2021

Triptolide targets super-enhancer networks in pancreatic cancer cells and cancer-associated fibroblasts.

Oncogenesis 2020 Nov 9;9(11):100. Epub 2020 Nov 9.

Molecular Medicine Division, Translational Genomics Research Institute, Phoenix, AZ, USA.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) is highly heterogeneous, fibrotic, and hypovascular, marked by extensive desmoplasia and maintained by the tumor cells, cancer-associated fibroblasts (CAFs) and other stromal cells. There is an urgent need to identify and develop treatment strategies that not only target the tumor cells but can also modulate the stromal cells. A growing number of studies implicate the role of regulatory DNA elements called super-enhancers (SE) in maintaining cell-type-specific gene expression networks in both normal and cancer cells. Using chromatin activation marks, we first mapped SE networks in pancreatic CAFs and epithelial tumor cells and found them to have distinct SE profiles. Next, we explored the role of triptolide (TPL), a natural compound with antitumor activity, in the context of modulating cell-type-specific SE signatures in PDAC. We found that TPL, cytotoxic to both pancreatic tumor cells and CAFs, disrupted SEs in a manner that resulted in the downregulation of SE-associated genes (e.g., BRD4, MYC, RNA Pol II, and Collagen 1) in both cell types at mRNA and protein levels. Our observations suggest that TPL acts as a SE interactive agent and may elicit its antitumor activity through SE disruption to re-program cellular cross talk and signaling in PDAC. Based on our findings, epigenetic reprogramming of transcriptional regulation using SE modulating compounds such as TPL may provide means for effective treatment options for pancreatic cancer patients.
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http://dx.doi.org/10.1038/s41389-020-00285-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653036PMC
November 2020

Single-cell transcriptome analysis of tumor and stromal compartments of pancreatic ductal adenocarcinoma primary tumors and metastatic lesions.

Genome Med 2020 09 29;12(1):80. Epub 2020 Sep 29.

Molecular Medicine Division, Translational Genomics Research Institute, 445 N. Fifth St., Phoenix, AZ, 85004, USA.

Background: Solid tumors such as pancreatic ductal adenocarcinoma (PDAC) comprise not just tumor cells but also a microenvironment with which the tumor cells constantly interact. Detailed characterization of the cellular composition of the tumor microenvironment is critical to the understanding of the disease and treatment of the patient. Single-cell transcriptomics has been used to study the cellular composition of different solid tumor types including PDAC. However, almost all of those studies used primary tumor tissues.

Methods: In this study, we employed a single-cell RNA sequencing technology to profile the transcriptomes of individual cells from dissociated primary tumors or metastatic biopsies obtained from patients with PDAC. Unsupervised clustering analysis as well as a new supervised classification algorithm, SuperCT, was used to identify the different cell types within the tumor tissues. The expression signatures of the different cell types were then compared between primary tumors and metastatic biopsies. The expressions of the cell type-specific signature genes were also correlated with patient survival using public datasets.

Results: Our single-cell RNA sequencing analysis revealed distinct cell types in primary and metastatic PDAC tissues including tumor cells, endothelial cells, cancer-associated fibroblasts (CAFs), and immune cells. The cancer cells showed high inter-patient heterogeneity, whereas the stromal cells were more homogenous across patients. Immune infiltration varies significantly from patient to patient with majority of the immune cells being macrophages and exhausted lymphocytes. We found that the tumor cellular composition was an important factor in defining the PDAC subtypes. Furthermore, the expression levels of cell type-specific markers for EMT cancer cells, activated CAFs, and endothelial cells significantly associated with patient survival.

Conclusions: Taken together, our work identifies significant heterogeneity in cellular compositions of PDAC tumors and between primary tumors and metastatic lesions. Furthermore, the cellular composition was an important factor in defining PDAC subtypes and significantly correlated with patient outcome. These findings provide valuable insights on the PDAC microenvironment and could potentially inform the management of PDAC patients.
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http://dx.doi.org/10.1186/s13073-020-00776-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523332PMC
September 2020

Ultra-Sensitive Automated Profiling of EpCAM Expression on Tumor-Derived Extracellular Vesicles.

Front Genet 2019 17;10:1273. Epub 2019 Dec 17.

Virginia G. Piper Biodesign Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, AZ, United States.

Extracellular vesicles (EVs) are abundant in most biological fluids and considered promising biomarker candidates, but the development of EV biomarker assays is hindered, in part, by their requirement for prior EV purification and the lack of standardized and reproducible EV isolation methods. We now describe a far-field nanoplasmon-enhanced scattering (FF-nPES) assay for the isolation-free characterization of EVs present in small volumes of serum (< 5 µl). In this approach, EVs are captured with a cancer-selective antibody, hybridized with gold nanorods conjugated with an antibody to the EV surface protein CD9, and quantified by their ability to scatter light when analyzed using a fully automated dark-field microscope system. Our results indicate that FF-nPES performs similarly to EV ELISA, when analyzing EV surface expression of epithelial cell adhesion molecule (EpCAM), which has clinical significant as a cancer biomarker. Proof-of-concept FF-nPES data indicate that it can directly analyze EV EpCAM expression from serum samples to distinguish early stage pancreatic ductal adenocarcinoma patients from healthy subjects, detect the development of early stage tumors in a mouse model of spontaneous pancreatic cancer, and monitor tumor growth in patient derived xenograft mouse models of pancreatic cancer. FF-nPES thus appears to exhibit strong potential for the direct analysis of EV membrane biomarkers for disease diagnosis and treatment monitoring.
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http://dx.doi.org/10.3389/fgene.2019.01273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928048PMC
December 2019

A DNA Polymerase Stop Assay for Characterization of G-Quadruplex Formation and Identification of G-Quadruplex-Interactive Compounds.

Methods Mol Biol 2019 ;2035:223-231

Molecular Medicine Division, Translational Genomics Research Institute, Phoenix, AZ, USA.

Guanine-rich DNA sequences are able to spontaneously fold into G-quadruplex structures in the presence of certain metal cations. In the human genome, the majority of DNA G-quadruplexes form at the telomeres and regulatory regions of cancer-related genes. The formation of these structures is implicated in nuclear processes involving DNA, including transcription, DNA replication, and DNA repair. In the past few decades, small molecules which can stabilize these structures have been shown to suppress the telomere extension and to inhibit oncogene transcription. Therefore, DNA G-quadruplexes are thought to be attractive targets for new anticancer therapies. In this chapter, we describe step by step a DNA polymerase extension method for the characterization of G-quadruplex formation and identification of G-quadruplex-interactive compounds. This method is based on the principle that DNA polymerase is incapable to resolve G-quadruplex structure and pauses at 3'-end of the G-quadruplex forming region when it transverses to the 5'-end of the template. Results from the DNA polymerase stop assay can provide the basis for further studies aimed at elucidating the major G-quadruplexes formed by sequences consisting of more than four runs of contiguous guanines, as well as the specificity of G-quadruplex-interactive molecules in binding different G-quadruplex topologies.
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http://dx.doi.org/10.1007/978-1-4939-9666-7_12DOI Listing
April 2020

Single-Cell Sequencing in Precision Medicine.

Cancer Treat Res 2019 ;178:237-252

Molecular Medicine Division, Translational Genomics Research Institute, Phoenix, AZ, USA.

The application of next-generation sequencing in cancer genomics allowed for a better understanding of the genetics and pathogenesis of cancer. Single-cell genomics is a relatively new field that has enhanced our current knowledge of the genetic diversity of cells involved in the complex biological systems of cancer. Single-cell genomics is a rapidly developing field, and current technologies can assay a single cell's gene expression, DNA variation, epigenetic state, and nuclear structure. Statistical and computational methods are central to single-cell genomics and allows for extraction of meaningful information. The translational application of single-cell sequencing in precision cancer therapy has the potential to improve cancer diagnostics, prognostics, targeted therapy, early detection, and noninvasive monitoring. Furthermore, single-cell genomics will transform cancer research as even initial experiments have revolutionized our current understanding of gene regulation and disease.
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http://dx.doi.org/10.1007/978-3-030-16391-4_9DOI Listing
June 2019

Triptolide and Its Derivatives as Cancer Therapies.

Trends Pharmacol Sci 2019 05 8;40(5):327-341. Epub 2019 Apr 8.

Molecular Medicine Division, Translational Genomics Research Institute, Phoenix, AZ, USA. Electronic address:

Triptolide, a compound isolated from a Chinese medicinal herb, possesses potent antitumor, immunosuppressive, and anti-inflammatory properties, but is clinically limited due to its poor solubility, bioavailability, and toxicity. Recently, Minnelide, a water-soluble prodrug of triptolide, was shown to have potent antitumor activity in various preclinical cancer models. Minnelide is currently in Phase II clinical trials for treatment of advanced pancreatic cancer, which has fueled increased interest in this promising agent. Here, we review the recent advances in the biological activity of triptolide and its analogs, their mechanisms of actions, and their clinical developments. A special emphasis is given to proteins and pathways within the tumor and stromal compartments that are targeted by triptolide and its analogs as well as the ongoing clinical trials.
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http://dx.doi.org/10.1016/j.tips.2019.03.002DOI Listing
May 2019

A Multiscale Map of the Stem Cell State in Pancreatic Adenocarcinoma.

Cell 2019 04 4;177(3):572-586.e22. Epub 2019 Apr 4.

Tumor Initiation and Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.

Drug resistance and relapse remain key challenges in pancreatic cancer. Here, we have used RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells, highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular, the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (RORγ), known to drive inflammation and T cell differentiation, was upregulated during pancreatic cancer progression, and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further, a large-scale retrospective analysis in patients revealed that RORγ expression may predict pancreatic cancer aggressiveness, as it positively correlated with advanced disease and metastasis. Collectively, these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells, suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients.
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http://dx.doi.org/10.1016/j.cell.2019.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711371PMC
April 2019

SuperCT: a supervised-learning framework for enhanced characterization of single-cell transcriptomic profiles.

Nucleic Acids Res 2019 05;47(8):e48

Translational Genomics Research Institute, Molecular Medicine Division, Phoenix, AZ, USA.

Characterization of individual cell types is fundamental to the study of multicellular samples. Single-cell RNAseq techniques, which allow high-throughput expression profiling of individual cells, have significantly advanced our ability of this task. Currently, most of the scRNA-seq data analyses are commenced with unsupervised clustering. Clusters are often assigned to different cell types based on the enriched canonical markers. However, this process is inefficient and arbitrary. In this study, we present a technical framework of training the expandable supervised-classifier in order to reveal the single-cell identities as soon as the single-cell expression profile is input. Using multiple scRNA-seq datasets we demonstrate the superior accuracy, robustness, compatibility and expandability of this new solution compared to the traditional methods. We use two examples of the model upgrade to demonstrate how the projected evolution of the cell-type classifier is realized.
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http://dx.doi.org/10.1093/nar/gkz116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486558PMC
May 2019

Chemoresistance Transmission via Exosome-Mediated EphA2 Transfer in Pancreatic Cancer.

Theranostics 2018 13;8(21):5986-5994. Epub 2018 Nov 13.

School of Biological and Health Systems Engineering, Virginia G. Piper Biodesign Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA.

Exosomes are small extracellular vesicles secreted by most cells that are found in blood and other bodily fluids, and which contain cytoplasmic material and membrane factors corresponding to their cell type of origin. Exosome membrane factors and contents have been reported to alter adjacent and distant cell behavior in multiple studies, but the impact of cancer-derived exosomes on chemoresistance is less clear. Exosomes isolated from three pancreatic cancer (PC) cell lines displaying variable gemcitabine (GEM) resistance (PANC-1, MIA PaCa-2, and BxPC-3) were tested for their capacity to transmit chemoresistance among these cell lines. Comparative proteomics was performed to identify key exosomal proteins that conferred chemoresistance. Cell survival was assessed in GEM responsive PC cell lines treated with recombinant Ephrin type-A receptor 2 (EphA2), a candidate chemoresistance transfer factor, or exosomes from a chemoresistant PC cell line treated with or without EphA2 shRNA. Exosomes from chemoresistant PANC-1 cells increased the GEM resistance of MIA PaCa-2 and BxPC-3 cell cultures. Comparative proteomics determined that PANC-1 exosomes overexpressed Ephrin type-A receptor 2 (EphA2) versus exosomes of less chemoresistant PC cell lines MIA PaCa-2 and BxPC-3. EphA2-knockdown in PANC-1 cells inhibited their ability to transmit exosome-mediated chemoresistance to MIA PaCa-2 and BxPC-3, while treatment of MIA PaCa-2 and BxPC-3 cells with soluble EphA2 did not promote chemoresistance, indicating that membrane carried EphA2 was important for the EphA2 chemoresistance effect. Exosomal EphA2 expression could transmit chemoresistance and may potentially serve as a minimally-invasive predictive biomarker for PC treatment response. Further work should address whether additional exosomal factors regulate resistance to other cancer therapeutic agents for PC or other cancer types.
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http://dx.doi.org/10.7150/thno.26650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299429PMC
September 2019

Dosage of Atg5 Gene Affects Pancreatic Tumorigenesis and Progression.

Authors:
Haiyong Han

Gastroenterology 2019 01 22;156(1):17-19. Epub 2018 Nov 22.

Molecular Medicine Division, Translational Genomics Research Institute, Phoenix, Arizona. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2018.11.038DOI Listing
January 2019

KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism.

Cancer Cell 2018 11;34(5):807-822.e7

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.
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http://dx.doi.org/10.1016/j.ccell.2018.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321749PMC
November 2018

The Effects of Synthetically Modified Natural Compounds on ABC Transporters.

Pharmaceutics 2018 Aug 9;10(3). Epub 2018 Aug 9.

School of Science, Auckland University of Technology, Auckland 1010, New Zealand.

Multidrug resistance (MDR) is a major hurdle which must be overcome to effectively treat cancer. ATP-binding cassette transporters (ABC transporters) play pivotal roles in drug absorption and disposition, and overexpression of ABC transporters has been shown to attenuate cellular/tissue drug accumulation and thus increase MDR across a variety of cancers. Overcoming MDR is one desired approach to improving the survival rate of patients. To date, a number of modulators have been identified which block the function and/or decrease the expression of ABC transporters, thereby restoring the efficacy of a range of anticancer drugs. However, clinical MDR reversal agents have thus far proven ineffective and/or toxic. The need for new, effective, well-tolerated and nontoxic compounds has led to the development of natural compounds and their derivatives to ameliorate MDR. This review evaluates whether synthetically modifying natural compounds is a viable strategy to generate potent, nontoxic, ABC transporter inhibitors which may potentially reverse MDR.
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http://dx.doi.org/10.3390/pharmaceutics10030127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161255PMC
August 2018

Parallel Accumulation of Tumor Hyaluronan, Collagen, and Other Drivers of Tumor Progression.

Clin Cancer Res 2018 10 3;24(19):4798-4807. Epub 2018 Jul 3.

Halozyme Therapeutics, Inc., San Diego, California.

The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors. We used immunohistochemistry, imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance. In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content. The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743334PMC
October 2018

Pixantrone: novel mode of action and clinical readouts.

Expert Rev Hematol 2018 07 18;11(7):587-596. Epub 2018 Jun 18.

d Istituto Oncologico di Ricerca , Università della Svizzera italiana , Bellinzona , Switzerland.

Introduction: Pixantrone is a first-in-class aza-anthracenedione approved as monotherapy for treatment of relapsed or refractory aggressive diffuse B-cell non-Hodgkin's lymphoma (NHL), a patient group which is notoriously difficult to treat. It has a unique chemical structure and pharmacologic properties distinguishing it from anthracyclines and anthracenediones. Areas covered: The chemical structure and mode of action of pixantrone versus doxorubicin and mitoxantrone; preclinical evidence for pixantrone's therapeutic effect and cardiac tolerability; efficacy and safety of pixantrone in clinical trials; ongoing and completed trials of pixantrone alone or as combination therapy; and the risk of cardiotoxicity of pixantrone versus doxorubicin and mitoxantrone. Expert commentary: Currently, pixantrone is the only approved therapy for multiply relapsed or refractory NHL, an area with few available effective treatment options. Pixantrone is currently being investigated as combination therapy with other drugs including several targeted therapies, with the ultimate goal of improved survival in heavily pretreated patients. In order for pixantrone to be acknowledged in the treatment of aggressive NHL, the perception of pixantrone as an anthracycline-like agent that has anthracycline-like activity and cardiotoxicity needs to be changed. Further data from ongoing clinical trials will help in confirming pixantrone as an effective and safe option.
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http://dx.doi.org/10.1080/17474086.2018.1476848DOI Listing
July 2018

Assessment of folate receptor alpha and beta expression in selection of lung and pancreatic cancer patients for receptor targeted therapies.

Oncotarget 2018 Jan 15;9(4):4485-4495. Epub 2017 Dec 15.

Department of Chemistry, Purdue University, West Lafayette, IN, USA.

A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-β. Logically, if the FR positive cell population in a tumor mass is dominated by FR-β positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-β, no study to date has investigated expression of both FR-α and FR-β in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-β (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-β combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-β while pancreatic cancers express primarily FR-β. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-β should be obtained to predict the potential efficacy of a folate-targeted drug.
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http://dx.doi.org/10.18632/oncotarget.23321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796989PMC
January 2018

RNA Interference to Knock Down Gene Expression.

Authors:
Haiyong Han

Methods Mol Biol 2018 ;1706:293-302

Translational Genomics Research Institute, 445. N. Fifth St., Suite 400, Phoenix, AZ, 85004, USA.

RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a tool for knocking down the expression of individual genes posttranscriptionally, RNAi has been widely used to study the cellular function of genes. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Potential challenges and problems associated with the siRNA technology are also discussed.
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http://dx.doi.org/10.1007/978-1-4939-7471-9_16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743327PMC
October 2018

Pancreatic acinar cell carcinoma: A review on molecular profiling of patient tumors.

World J Gastroenterol 2017 Dec;23(45):7945-7951

Molecular Medicine Division, Translational Genomics Research Institute (TGen), Phoenix, AZ 85004, United States.

Pancreatic carcinomas with acinar differentiation are rare, accounting for 1%-2% of adult pancreatic tumors; they include pancreatic acinar cell carcinoma (PACC), pancreatoblastoma, and carcinomas of mixed differentiation. Patients with PACC have a prognosis better than pancreatic ductal adenocarcinomas but worse than pancreatic neuroendocrine tumors. Reports of overall survival range from 18 to 47 mo. A literature review on PACCs included comprehensive genomic profiling and whole exome sequencing on a series of more than 70 patients as well as other diagnostic studies including immunohistochemistry. Surgical resection of PACC is the preferred treatment for localized and resectable tumors. The efficacy of adjuvant treatment is unclear. Metastatic PACCs are generally not curable and treated with systemic chemotherapy. They are moderately responsive to chemotherapy with different regimens showing various degrees of response in case reports/series. Most of these regimens were developed to treat patients with pancreatic ductal adenocarcinomas or colorectal adenocarcinomas. Review of PACC's molecular profiling showed a number of gene alterations such as: , , , , , , , , , and DNA mismatch repair abnormalities. PACCs had multiple somatic mutations with some targetable with available drugs. Therefore, molecular profiling of PACC should be an option for patients with refractory PACC.
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http://dx.doi.org/10.3748/wjg.v23.i45.7945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725289PMC
December 2017

Curcumin sensitizes pancreatic cancer cells to gemcitabine by attenuating PRC2 subunit EZH2, and the lncRNA PVT1 expression.

Carcinogenesis 2017 10;38(10):1036-1046

Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott and White Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX 75246, USA.

Development of resistance to chemotherapeutic drugs is a major challenge in the care of patients with pancreatic ductal adenocarcinoma (PDAC). Acquired resistance to chemotherapeutic agents in PDAC has been linked to a subset of cancer cells termed 'cancer stem cells' (CSCs). Therefore, an improved understanding of the molecular events underlying the development of pancreatic CSCs is required to identify new therapeutic targets to overcome chemoresistance. Accumulating evidence indicates that curcumin, a phenolic compound extracted from turmeric, can overcome de novo chemoresistance and re-sensitize tumors to various chemotherapeutic agents. However, the underlying mechanisms for curcumin-mediated chemosensitization remain unclear. The Enhancer of Zeste Homolog-2 (EZH2) subunit of Polycomb Repressive Complex 2 (PRC2) was recently identified as a key player regulating drug resistance. EZH2 mediates interaction with several long non-coding RNAs (lncRNAs) to modulate epithelial-mesenchymal transition and cancer stemness, phenomena commonly associated with drug resistance. Here, we report the re-sensitization of chemoresistant PDAC cells by curcumin through the inhibition of the PRC2-PVT1-c-Myc axis. Using gemcitabine-resistant PDAC cell lines, we found that curcumin sensitized chemoresistant cancer cells by inhibiting the expression of the PRC2 subunit EZH2 and its related lncRNA PVT1. Curcumin was also found to prevent the formation of spheroids, a hallmark of CSCs, and to down-regulate several self-renewal driving genes. In addition, we confirmed our in vitro findings in a xenograft mouse model where curcumin inhibited gemcitabine-resistant tumor growth. Overall, this study indicates clinical relevance for combining curcumin with chemotherapy to overcome chemoresistance in PDAC.
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http://dx.doi.org/10.1093/carcin/bgx065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862331PMC
October 2017

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts.

J Vis Exp 2017 08 23(126). Epub 2017 Aug 23.

Molecular Medicine Division, Translational Genomics Research Institute;

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 µm within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.
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http://dx.doi.org/10.3791/56081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614363PMC
August 2017

Inhibition of ROCK1 kinase modulates both tumor cells and stromal fibroblasts in pancreatic cancer.

PLoS One 2017 25;12(8):e0183871. Epub 2017 Aug 25.

Molecular Medicine Division, The Translational Genomics Research Institute, Phoenix, Arizona, United States of America.

ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC50s of 6-71 μM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183871PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571985PMC
October 2017

Clinical study of genomic drivers in pancreatic ductal adenocarcinoma.

Br J Cancer 2017 Aug 18;117(4):572-582. Epub 2017 Jul 18.

Translational Genomics Research Institute, Phoenix, AZ 85004, USA.

Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials.

Methods: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed.

Results: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection.

Conclusions: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.
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http://dx.doi.org/10.1038/bjc.2017.209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558689PMC
August 2017

Blocking Nerve Growth Factor Signaling Reduces the Neural Invasion Potential of Pancreatic Cancer Cells.

PLoS One 2016 28;11(10):e0165586. Epub 2016 Oct 28.

Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, United States of America.

Perineural invasion (PNI) is thought to be one of the factors responsible for the high rate of tumor recurrence after surgery and the pain generation associated with pancreatic cancer. Signaling via the nerve growth factor (NGF) pathway between pancreatic cancer cells and the surrounding nerves has been implicated in PNI, and increased levels of these proteins have been correlated to poor prognosis. In this study, we examine the molecular mechanism of the NGF signaling pathway in PNI in pancreatic cancer. We show that knocking down NGF or its receptors, TRKA and p75NTR, or treatment with GW441756, a TRKA kinase inhibitor, reduces the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, pancreatic cancer cells migrate towards dorsal root ganglia (DRG) in a co-culture assay, indicating a paracrine NGF signaling between the DRGs and pancreatic cancer cells. Knocking down the expression of NGF pathway proteins or inhibiting the activity of TRKA by GW441756 reduced the migratory ability of Mia PaCa2 towards the DRGs. Finally, blocking NGF signaling by NGF neutralizing antibodies or GW441756 inhibited the neurite formation in PC-12 cells in response to conditioned media from pancreatic cancer cells, indicating a reciprocal signaling pathway between the pancreatic cancer cells and nerves. Our results indicate that NGF signaling pathway provides a potential target for developing molecularly targeted therapies to decrease PNI and reduce pain generation. Since there are several TRKA antagonists currently in early clinical trials they could now be tested in the clinical situation of pancreatic cancer induced pain.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085053PMC
June 2017

Adenosquamous carcinoma of the pancreas: Molecular characterization of 23 patients along with a literature review.

World J Gastrointest Oncol 2015 Sep;7(9):132-40

Erkut Borazanci, Daniel D Von Hoff, HonorHealth Research Institute, Virginia G. Piper Cancer Center, Scottsdale, AZ 85258, United States.

Adenosquamous carcinoma of the pancreas (ASCP) is a rare entity. Like adenocarcinoma of the pancreas, overall survival is poor. Characteristics of ASCP include central tumor necrosis, along with osteoclasts and hypercalcemia. Various theories exist as to why this histological subtype exists, as normal pancreas tissue has no benign squamous epithelium. Due to the rarity of this disease, limited molecular analysis has been performed, and those reports indicate unique molecular features of ASCP. In this paper, we characterize 23 patients diagnosed with ASCP through molecular profiling using immunohistochemistry staining, fluorescent in situ hybridization, chromogenic in situ hybridization, and gene sequencing, Additionally, we provide a comprehensive literature review of what is known to date of ASCP. Molecular characterization revealed overexpression in MRP1 (80%), MGMT (79%), TOP2A (75), RRM1 (42%), TOPO1 (42%), PTEN (45%), CMET (40%), and C-KIT (10%) among others. One hundred percent of samples tested were positive for KRAS mutations. This analysis shows heretofore unsuspected leads to be considered for treatments of this rare type of exocrine pancreas cancer. Molecular profiling may be appropriate to provide maximum information regarding the patient's tumor. Further work should be pursued to better characterize this disease.
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http://dx.doi.org/10.4251/wjgo.v7.i9.132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569590PMC
September 2015

Orchestrating the Tumor Microenvironment to Improve Survival for Patients With Pancreatic Cancer: Normalization, Not Destruction.

Cancer J 2015 Jul-Aug;21(4):299-306

From the Clinical Translational Research Division, The Translational Genomics Research Institute (TGen), Phoenix, AZ.

Pancreatic cancer is the fourth leading cause of cancer death in the United States. The microenvironment of pancreatic cancer could be one of the "perfect storms" that support the growth of a cancer. Indeed, pancreatic cancer may be the poster child of a problem with the microenvironment. In this article, we review the rationale and attempts to date on modifying or targeting structural proteins in the microenvironment including hyaluronan (HA) (in primary and metastases), collagen, and SPARC (secreted protein, acidic, and rich in cysteine). Indeed, working in this area has produced a regimen that improves survival for patients with advanced pancreatic cancer (nab-paclitaxel + gemcitabine). In addition, in initial clinical trials, PEGylated hyaluronidase appears promising. We also review a new approach that is different than targeting/destroying the microenvironment and that is orchestrating, reengineering, reprogramming, or normalizing the microenvironment (including normalizing structural proteins, normalizing an immunologically tumor-friendly environment to a less friendly environment, reversing epithelial-to-mesenchymal transition, and so on). We believe this will be most effectively done by agents that have global effects on transcription. There is initial evidence that this can be done by agents such as vitamin D derivatives and other new agents. There is no doubt these opportunities can now be tried in the clinic with hopefully beneficial effects.
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http://dx.doi.org/10.1097/PPO.0000000000000140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817719PMC
May 2016

Desmoplasia in Primary Tumors and Metastatic Lesions of Pancreatic Cancer.

Clin Cancer Res 2015 Aug 18;21(15):3561-8. Epub 2015 Feb 18.

Clinical Translational Research Division, The Translational Genomics Research Institute, Phoenix, Arizona.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is characterized by high levels of fibrosis, termed desmoplasia, which is thought to hamper the efficacy of therapeutics treating PDAC. Our primary focus was to evaluate differences in the extent of desmoplasia in primary tumors and metastatic lesions. As metastatic burden is a primary cause for mortality in PDAC, the extent of desmoplasia in metastases may help to determine whether desmoplasia targeting therapeutics will benefit patients with late-stage, metastatic disease.

Experimental Design: We sought to assess desmoplasia in metastatic lesions of PDAC and compare it with that of primary tumors. Fifty-three patients' primaries and 57 patients' metastases were stained using IHC staining techniques.

Results: We observed a significant negative correlation between patient survival and extracellular matrix deposition in primary tumors. Kaplan-Meier curves for collagen I showed median survival of 14.6 months in low collagen patients, and 6.4 months in high-level patients (log rank, P < 0.05). Low-level hyaluronan patients displayed median survival times of 24.3 months as compared with 9.3 months in high-level patients (log rank, P < 0.05). Our analysis also indicated that extracellular matrix components, such as collagen and hyaluronan, are found in high levels in both primary tumors and metastatic lesions. The difference in the level of desmoplasia between primary tumors and metastatic lesions was not statistically significant.

Conclusions: Our results suggest that both primary tumors and metastases of PDAC have highly fibrotic stroma. Thus, stromal targeting agents have the potential to benefit PDAC patients, even those with metastatic disease.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-1051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526394PMC
August 2015

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

PLoS Genet 2014 Feb 13;10(2):e1004135. Epub 2014 Feb 13.

Translational Genomics Research Institute, Phoenix, Arizona, United States of America.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.
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http://dx.doi.org/10.1371/journal.pgen.1004135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923676PMC
February 2014

Therapeutic melting pot of never in mitosis gene a related kinase 2 (Nek2): a perspective on Nek2 as an oncology target and recent advancements in Nek2 small molecule inhibition.

J Med Chem 2014 Jul 25;57(14):5835-44. Epub 2014 Feb 25.

Department of Pharmacoloy and Toxicology, College of Pharmacy, The University of Arizona , Tucson, Arizona 85721, United States.

The global incidence of cancer is on the rise, and within the next decade, the disease is expected to become the leading cause of death worldwide. Forthcoming strategies used to treat cancers focus on the design and implementation of multidrug therapies to target complementary cancer specific pathways. A more direct means by which this multitargeted approach can be achieved is by identifying and targeting interpathway regulatory factors. Recent advances in understanding Nek2 (NIMA related kinase 2) biology suggest that the kinase potentially represents a multifaceted therapeutic target. In this regard, pharmacologic modulation of Nek2 with a single agent may effect several mechanisms important for tumor growth, survival, progression, and metastasis. We herein review the development of Nek2 as an oncology target and provide a succinct chronology of drug discovery campaigns focused on targeting Nek2.
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http://dx.doi.org/10.1021/jm401719nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666018PMC
July 2014

Discrimination between adenocarcinoma and normal pancreatic ductal fluid by proteomic and glycomic analysis.

J Proteome Res 2014 Feb 12;13(2):395-407. Epub 2013 Dec 12.

Complex Carbohydrate Research Center, ‡Chemistry Department, and §Department of Biochemistry and Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.

Sensitive and specific biomarkers for pancreatic cancer are currently unavailable. The high mortality associated with adenocarcinoma of the pancreatic epithelium justifies the broadest possible search for new biomarkers that can facilitate early detection or monitor treatment efficacy. Protein glycosylation is altered in many cancers, leading many to propose that glycoproteomic changes may provide suitable biomarkers. In order to assess this possibility for pancreatic cancer, we have performed an in-depth LC-MS/MS analysis of the proteome and MS(n)-based characterization of the N-linked glycome of a small set of pancreatic ductal fluid obtained from normal, pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma patients. Our results identify a set of seven proteins that were consistently increased in cancer ductal fluid compared to normal (AMYP, PRSS1, GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was consistently decreased (LIPR2). These proteins are all directly or indirectly associated with the secretory pathway in normal pancreatic cells. Validation of these changes in abundance by Western blotting revealed increased REG protein glycoform diversity in cancer. Characterization of the total N-linked glycome of normal, IPMN, and adenocarcinoma ductal fluid clustered samples into three discrete groups based on the prevalence of six dominant glycans. Within each group, the profiles of less prevalent glycans were able to distinguish normal from cancer on this small set of samples. Our results emphasize that individual variation in protein glycosylation must be considered when assessing the value of a glycoproteomic marker, but also indicate that glycosylation diversity across human subjects can be reduced to simpler clusters of individuals whose N-linked glycans share structural features.
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http://dx.doi.org/10.1021/pr400422gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946306PMC
February 2014

Isolation and characterization of muscle fatigue substance with anti-tumor activities.

J Cancer 2013 9;4(4):343-9. Epub 2013 May 9.

Division of Clinical Translational Research, Translational Genomics Research Institute, Scottsdale, Arizona, USA.

Research during the 1950's indicated that exercise played a role in the reduction of tumor growth. In the 1960's our studies confirmed that tumor-bearing rats, exercised to fatigue, demonstrated tumor inhibition. Our further studies isolated an extract (Fatigue Substance, or F-Substance) from rectus femoris muscles of rats which had been electrically stimulated to fatigue. This extract significantly inhibited growth of transplanted rat tumors. Research continued until 1978 when it became apparent the methodology at that time was not able to further identify the substance's active components. Using current technology, we now report on the further isolation and characterization of F-Substance. In cell proliferation assays, extracts from electrically stimulated rat rectus femoris muscles had more significant inhibitory effect on the breast cancer cell line MCF-7 than those isolated from unstimulated muscles. To identify the molecule(s) responsible for the antitumor activity, a rat cytokine antibody array was used to profile the cytokines in the substances. Among the 29 different cytokines contained on the array, 3 showed greater than 3-fold difference between the substances isolated from the stimulated and unstimulated muscles. LIX (also known as CXCL5) is 6-fold higher in the substances isolated from stimulated muscles than those from the unstimulated muscles. TIMP-1 is 4.6 fold higher and sICAM is 3.6 fold higher in the substances from the stimulated muscles. Our results indicated that cytokines released from contracting muscles might be responsible for the antitumor effect of F-Substance.
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http://dx.doi.org/10.7150/jca.5418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654491PMC
May 2013