Bedabrata Sarkar

c-Met is a marker of pancreatic cancer stem cells and therapeutic target.

BACKGROUND & AIMS Growth of many different tumor types requires a population of self-renewing cancer stem cells (CSCs). c-Met is a marker of normal mouse pancreatic stem and progenitor cells; we investigated whether it is also a marker of human pancreatic CSCs that might be developed as a therapeutic target. METHODS We studied growth of primary human pancreatic adenocarcinoma in NOD SCID mice. The self-renewal capability of pancreatic cancer cells that expressed high levels of c-Met (c-Met(high)) was assessed using in vitro sphere assays and compared with those that were c-Met negative or expressed low levels of c-Met. The tumorigenicity of c-Met(high) pancreatic cancer cells was evaluated in NOD SCID mice. RESULTS c-Met(high) cells readily formed spheres, whereas c-Met-negative cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with small hairpin RNAs significantly inhibited tumor sphere formation. c-Met(high) cells had increased tumorigenic potential in mice; those that expressed c-Met and CD44 (0.5%-5% of the pancreatic cancer cells) had the capability for self-renewal and the highest tumorigenic potential of all cell populations studied. In pancreatic tumors established in NOD SCID mice, c-Met inhibitors slowed tumor growth and reduced the population of CSCs when given alone or in combination with gemcitabine. Administration of XL184 for 2 weeks after cardiac injection of cancer cells prevented the development of metastases. CONCLUSIONS c-Met is a new marker for pancreatic CSCs. It is required for growth and metastasis of pancreatic tumors in mice and is a therapeutic target for pancreatic cancer.

Selective Degradation of AU-Rich mRNAs Promoted by the p37 AUF1 Protein Isoform

ABSTRACT An AU-rich element (ARE) consisting of repeated canonical AUUUA motifs confers rapid degradation to many cytokine mRNAs when present in the 3′ untranslated region. Destabilization of mRNAs with AREs (ARE-mRNAs) is consistent with the interaction of ARE-binding proteins such as tristetraprolin and the four AUF1 isoforms. However, the association of the AUF1-mRNA interaction with decreased ARE-mRNA stability is correlative and has not been directly tested. We therefore determined whether overexpression of AUF1 isoforms promotes ARE-mRNA destabilization and whether AUF1 isoforms are limiting components for ARE-mRNA decay. We show that the p37 AUF1 isoform and, to a lesser extent, the p40 isoform possess ARE-mRNA-destabilizing activity when overexpressed. Surprisingly, overexpressed p37 AUF1 also destabilized reporter mRNAs containing a noncanonical but AU-rich 3′ untranslated region. Since overexpressed p37 AUF1 could interact in vivo with the AU-rich reporter mRNA, AUF1 may be involved in rapid turnover of mRNAs that lack canonical AREs. Moreover, overexpression of p37 AUF1 restored the ability of cells to rapidly degrade ARE-mRNAs when that ability was saturated and inhibited by overexpression of ARE-mRNAs. Finally, activation of ARE-mRNA decay often involves a translation-dependent step, which was eliminated by overexpression of p37 AUF1. These data indicate that the p37 AUF1 isoform and, to some extent, the p40 isoform are limiting factors that facilitate rapid decay of AU-rich mRNAs.

Nutritional Control of mRNA Stability Is Mediated by a Conserved AU-rich Element That Binds the Cytoplasmic Shuttling Protein HuR

The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the distal 217 bases of the 3′-untranslated region. When this 217-nucleotide nutrient sensorAU-rich element (NS-ARE) is present in a chimeric mRNA it confers mRNA stabilization during amino acid starvation. HuR is a member of the ELAV family of RNA-binding proteins that has been implicated in regulating the stability of ARE-containing mRNAs. We show here that the cytoplasmic concentration of HuR increases during amino acid starvation, at a time when total cellular HuR levels decrease. In addition, RNA gel shift experiments in vitro demonstrated that HuR binds to the NS-ARE and binding was dependent on the 11 residue AU-rich element. Moreover, HuR binding to the NS-ARE in extracts from amino acid-starved cells increased in parallel with the accumulation of cytoplasmic HuR. It is proposed that an adaptive response of cells to nutritional stress results in increased mRNA stability mediated by HuR binding to the NS-ARE.

Ubiquitin-dependent mechanism regulates rapid turnover of AU-rich cytokine mRNAs

An AU rich element (ARE) in the 3′ noncoding region promotes the rapid degradation of mammalian cytokine and proto-oncogene mRNAs, such as tumor necrosis factor-α, granulocyte–macrophage colony-stimulating factor (GM-CSF) and c-fos. Destabilization of ARE-mRNAs involves the association of ARE-binding proteins tristetraprolin or AUF1 and proteasome activity, of which the latter has not been characterized. Here, we show that the stability of a model short-lived mRNA containing the GM-CSF ARE was regulated by the level of ubiquitin-conjugating activity in the cell, which links ARE-mRNA decay to proteasome activity. Increased expression of a cytokine-inducible deubiquitinating protein (DUB) that impairs addition of ubiquitin to proteins fully blocked ARE-mRNA decay, whereas increased expression of a DUB that promotes ubiquitin addition to proteins strongly accelerated ARE-mRNA decay. ARE-mRNA turnover was found to be activated by the ubiquitin-addition reaction and blocked by the ubiquitin-removal reaction. Saturation of the ARE-mRNA decay machinery by high levels of ARE-mRNA, which is well established but not understood, was found to be relieved by increased expression of a DUB that promotes ubiquitin addition to proteins. Finally, inhibition of proteasome activity also blocked accelerated ARE-mRNA decay that is mediated by increased ubiquitin recycling. These results demonstrate that both ubiquitinating activity and proteasome activity are essential for rapid turnover of a model cytokine ARE-mRNA containing the GM-CSF ARE.

American College of Surgeons' Committee on Trauma Performance Improvement and Patient Safety Program: Maximal Impact in a Mature Trauma Center

BACKGROUND To examine the impact of an ongoing comprehensive performance improvement and patient safety (PIPS) program implemented in 2005 on mortality outcomes for trauma patients at an established American College of Surgeons (ACS)-verified Level I Trauma Center. METHODS The primary outcome measure was in-hospital mortality. Age, Injury Severity Score (ISS), and intensive care unit admissions were used as stratifying variables to examine outcomes over a 5-year period (2004-2008). Institution mortality rates were compared with the National Trauma Data Bank mortality rates stratified by ISS score. Enhancements to our comprehensive PIPS program included revision of trauma activation criteria, development of standardized protocols for initial resuscitation, massive transfusion, avoidance of over-resuscitation, tourniquet use, pelvic fracture management, emphasis on timely angiographic and surgical intervention, prompt spine clearance, reduction in time to computed tomography imaging, reduced dwell time in emergency department, evidence-based traumatic brain injury management, and multidisciplinary efforts to reduce healthcare-associated infections. RESULTS In 2004 (baseline data), the in-hospital mortality rate for the most severely injured trauma patients (ISS >24) at our trauma center was 30%, consistent with the reported mortality rate from the National Trauma Data Bank for patients with this severity of injury. Over 5 years, our mortality rate decreased significantly for severely injured patients with an ISS >24, from 30.1% (2004) to 18.3% (2008), representing a 12% absolute reduction in mortality (p = 0.011). During the same 5-year time period, the proportion of elderly patients (age >65 years) cared for at our trauma center increased from 23.5% in 2004 to 30.6% in 2008 (p = 0.0002). Class I trauma activations increased significantly from 5.5% in 2004 to 15.5% in 2008 based on our reclassification. A greater percentage of patients were admitted to the intensive care unit (25.8% in 2004 to 37.3% in 2007 and 30.4% in 2008). No difference was identified in the rate of blunt (95%) or penetrating (5%) mechanism of injury in our patients over this time period. Trauma Quality Improvement Program confirmed improved trauma outcomes with observed-to-expected ratio and 95% confidence intervals of 0.64 (0.42-0.86) for all patients, 0.54 (0.15-0.91) for blunt single-system patients, and 0.78 (0.51-1.06) for blunt multisystem patients. CONCLUSION Implementation of a multifaceted trauma PIPS program aimed at improving trauma care significantly reduced in-hospital mortality in a mature ACS Level I trauma center. Optimal care of the injured patient requires uncompromising commitment to PIPS.

Cancer stem cells: a new theory regarding a timeless disease.

Although cancer has been described in early medical texts from antiquity, it remains the second leading cause of death in the United States. Technological improvements in screening modalities have increased detection of smaller tumors, yet current therapies for most types of cancer often fail. Cancer death is usually attributable to the development of metastatic disease. Chemotherapeutic regimens target all proliferating cells based on the principle that tumor cells proliferate at a faster rate than normal cells, resulting in differential cytotoxicity. The increased proliferative capacity of cancer cells is the result of accumulated genetic insults to various cellular pathways. These mutations include those that enhance cellular proliferation or suppress normal growth inhibitory mechanisms and apoptosis. Still other mutations allow tumor cells to evade surveillance and removal by the immune system. Cumulatively, these mutations result in neoplastic tumor growth and subsequent distant metastasis. The tumor microenvironment has also emerged as a critical component in the development of cancer from benign neoplasia, both in secreted factors that modulate tumor cells and in three-dimensional interactions with extracellular matrix proteins. A paradigm shift in cancer biologists’ thinking about solid organ tumors may provide a new understanding of cancer development and progression and has implications for how we think about developing therapies to treat cancer patients. In normal tissues and organs, stem cells reside at the apex of a hierarchical scheme that drives organogenesis. The realization that tumors themselves function like complex organs birthed the theory that cancer cells with the properties of stem cells may be the key drivers of the complex machinery behind tumorigenesis. The cancer stem cell theory is based on the finding that a small number of highly tumorigenic cells within a tumor can produce the heterogeneous populations found within an entire tumor when transplanted into immunocompromised mice. Thus, it is believed that cancer stem cells (CSCs) may be responsible for tumor formation based upon their capacity for selfrenewal and differentiation. These capacities together permit asymmetric division of the stem cell, resulting in maintenance of the parental stem cell population in addition to production of differentiated progeny (Figure 1). The CSC population can be serially transplanted without loss of tumorigenicity due to its ability to undergo self-renewal. Given these findings, many have hypothesized that CSCs arise from genetic mutations that occur in normal stem cells. Normal stem cells possess the longest life span among mammalian cells, and it is thought that these cells are more likely to accumulate mutations over time and ultimately assume a malignant phenotype. Mutation of normal stem cells may create a population of CSCs that cause tumor growth as a result of altered self-renewal mechanisms, although specific mutations have yet to be demonstrated. Recent data supports such a model for colon cancer development. Barker et al. utilized a mouse model where the adenomatous polyposis coli (APC) gene of the Wnt signaling pathway was conditionally inactivated in the intestinal stem cell.1 These stem cells are located at the bottom of the intestinal crypt in both the small intestine and colon and become transformed with APC deletion within days. The transformed stem cells go on to form adenomas, in contrast to APC deletion in the progeny of the stem cells where adenomas rarely form. Alternatively, some studies suggest that CSCs may arise from mutated progenitor cells called transit amplifying cells that develop the capacity for unregulated self-renewal.2,3 Yet in other models, CSCs may arise from differentiated cells that assume a stem-cell phenotype. For example, in genetically engineered mouse models of pancreatic cancer, one study has suggested that the cell of origin of pancreatic cancer is an acinar or centroacinar cell.4 Additional work is being performed in several laboratories to verify this finding. Gene profiling experiments comparing CSCs and nonCSCs have revealed upregulation of other signaling pathways that are also found to be abnormal in human tumor specimens. These include Wnt/ -catenin, Notch, PI3KinaseAkt-mTOR, as well as the Hedgehog signaling pathway. For example, c-Myc, a downstream target of the -catenin signaling pathway, was found to be upregulated in glioma * To whom correspondence should be addressed. Mailing address: Depts. of Surgery and Molecular and Integrative Physiology, TC 2210B, Box 5343, University of Michigan Medical Center, 1500 E. Medical Center Dr., Ann Arbor, MI 48109. Phone: (734) 615-1600. Fax: (734) 936-5830, E-mail: simeone@umich.edu. † Department of Surgery. ‡ Department of Molecular and Integrative Physiology. Chem. Rev. 2009, 109, 3200–3208 3200

Human thymus mesenchymal stromal cells augment force production in self-organized cardiac tissue

BACKGROUND Mesenchymal stromal cells have been recently isolated from thymus gland tissue discarded after surgical procedures. The role of this novel cell type in heart regeneration has yet to be defined. The purpose of this study was to evaluate the therapeutic potential of human thymus-derived mesenchymal stromal cells using self-organized cardiac tissue as an in vitro platform for quantitative assessment. METHODS Mesenchymal stromal cells were isolated from discarded thymus tissue from neonates undergoing heart surgery and were incubated in differentiation media to demonstrate multipotency. Neonatal rat cardiomyocytes self-organized into cardiac tissue fibers in a custom culture dish either alone or in combination with varying numbers of mesenchymal stromal cells. A transducer measured force generated by spontaneously contracting self-organized cardiac tissue fibers. Work and power outputs were calculated from force tracings. Immunofluorescence was performed to determine the fate of the thymus-derived mesenchymal stromal cells. RESULTS Mesenchymal stromal cells were successfully isolated from discarded thymus tissue. After incubation in differentiation media, mesenchymal stromal cells attained the expected phenotypes. Although mesenchymal stromal cells did not differentiate into mature cardiomyocytes, addition of these cells increased the rate of fiber formation, force production, and work and power outputs. Self-organized cardiac tissue containing mesenchymal stromal cells acquired a defined microscopic architecture. CONCLUSIONS Discarded thymus tissue contains mesenchymal stromal cells, which can augment force production and work and power outputs of self-organized cardiac tissue fibers by several-fold. These findings indicate the potential utility of mesenchymal stromal cells in treating heart failure.

Abstract LB-257: c-Met: a new cancer stem cell marker and therapeutic target for pancreatic cancer

Pancreatic adenocarcinoma is a highly aggressive disease which is usually diagnosed in an advanced state and for which there are little/no effective therapies. The growth of many cancers depends on a subpopulation of self-renewing cells, termed cancer stem cells (CSC). We have previously identified such a CSC population in human pancreatic cancers which possess the cell surface marker phenotype CD44 + CD24 + ESA + . C-Met, a member of the receptor tyrosine kinases (RTKs) family and receptor for the hepatocyte growth factor (HGF) ligand, has been recently described as a marker of normal mouse pancreatic stem/progenitor cells. We hypothesized that c-Met may function as a cell surface marker and therapeutic target for human pancreatic CSCs. Studies were performed in low passage primary human pancreatic adenocarcinoma xenografts (n = 3) established in NOD-SCID mice. Self-renewal capacity of c-Met high pancreatic cancer cells was assessed using in vitro sphere assays and in vivo tumorigenicity of c-Met high pancreatic cancer cells was evaluated by cell implantation in NOD/SCID mice. In vitro studies demonstrated that c-Met high cells formed spheres in non-adherent cultures, while c-Met − cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with shRNA significantly inhibited tumor sphere formation. Pancreatic cancer cells expressing high c-Met had increased tumorigenic potential in vivo and cells expressing both CD44 and c-Met high , representing 0.5-5% of pancreatic cancer cells, demonstrated the highest tumorigenic potential of all populations studied to date. C-Met high CD44+ expressing pancreatic cancer cells demonstrated the capacity for self-renewal and production of differentiated progeny, properties that define CSCs. Further, c-Met inhibition of established primary human pancreatic tumors in NOD/SCID mice significantly inhibited tumor growth and depleted the pancreatic CSC population, when used alone or in combination with gemcitabine. Inhibition of c-Met also prevented the developmental of metastases using an intracardiac injection model. In this report, we define c-Met as a new marker for pancreatic CSC and define c-Met hi g h CD44 + cells as a highly tumorigenic pancreatic CSC population. Our data also indicate that targeting c-Met may be useful in eradicating a CSC population and inhibiting the development of metastases. 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-257.