Mathieu C. Rousseau

LPS-Induced TLR4 Signaling in Human Colorectal Cancer Cells Increases β1 Integrin-Mediated Cell Adhesion and Liver Metastasis

Infectious complications resulting from resection of colorectal cancer (CRC) elevates the risk of cancer recurrence and metastasis, but the reason for this risk relationship is unknown. Defining the mechanisms responsible may offer opportunities to improve outcomes in a majority of patients whose tumors are resected as part of their therapy. The complex formed between Toll receptor TLR4 and myeloid differentiation factor MD2 defines a major cell surface receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen that has been implicated in infectious complications after CRC resection. As the TLR4/MD2 complex is expressed on CRC cells, we hypothesized that LPS may promote liver metastasis in CRC by stimulating TLR4 signaling. In support of this hypothesis, we report here that LPS enhances liver metastasis of human CRC cells that express TLR4/MD2 after intrasplenic graft of immunocompromised nude mice. Compared with TLR4 nonexpressing, nonmetastatic CRC cells, we observed increased in vitro adherence to different extracellular matrices and human umbilical vein endothelial cells (HUVEC). Furthermore, we observed an increased likelihood of in vivo capture within hepatic sinusoids after LPS treatment. No differences were apparent in phosphorylation of p38 and MAPK isoforms, but in metastatic CRC cells expressing surface TLR4 treatment with LPS increased Ser473 phosphorylation of AKT kinase. We showed that enhanced adherence elicited by LPS in these cells could be blocked at three different levels, using Eritoran (TLR4 small molecule antagonist), PI-103 (PI3K inhibitor), or anti-β1 integrin blocking antibodies. Taken together, the results indicate that stimulation of the TLR4/MD2 complex by LPS activates PI3K/AKT signaling and promotes downstream β1 integrin function, thereby increasing the adhesiveness and metastatic capacity of CRC cells. Our findings suggest that inhibiting LPS-induced TLR4 signaling could improve therapeutic outcomes by preventing cancer metastasis during the perioperative period of CRC resection.

The three-dimensional architecture of Hox cluster silencing

Spatial chromatin organization is emerging as an important mechanism to regulate the expression of genes. However, very little is known about genome architecture at high-resolution in vivo. Here, we mapped the three-dimensional organization of the human Hox clusters with chromosome conformation capture (3C) technology. We show that computational modeling of 3C data sets can identify candidate regulatory proteins of chromatin architecture and gene expression. Hox genes encode evolutionarily conserved master regulators of development which strict control has fascinated biologists for over 25 years. Proper transcriptional silencing is key to Hox function since premature expression can lead to developmental defects or human disease. We now show that the HoxA cluster is organized into multiple chromatin loops that are dependent on transcription activity. Long-range contacts were found in all four silent clusters but looping patterns were specific to each cluster. In contrast to the Drosophila homeotic bithorax complex (BX-C), we found that Polycomb proteins are only modestly required for human cluster looping and silencing. However, computational three-dimensional Hox cluster modeling identified the insulator-binding protein CTCF as a likely candidate mediating DNA loops in all clusters. Our data suggest that Hox cluster looping may represent an evolutionarily conserved structural mechanism of transcription regulation.

Three-dimensional modeling of chromatin structure from interaction frequency data using Markov chain Monte Carlo sampling

BackgroundLong-range interactions between regulatory DNA elements such as enhancers, insulators and promoters play an important role in regulating transcription. As chromatin contacts have been found throughout the human genome and in different cell types, spatial transcriptional control is now viewed as a general mechanism of gene expression regulation. Chromosome Conformation Capture Carbon Copy (5C) and its variant Hi-C are techniques used to measure the interaction frequency (IF) between specific regions of the genome. Our goal is to use the IF data generated by these experiments to computationally model and analyze three-dimensional chromatin organization.ResultsWe formulate a probabilistic model linking 5C/Hi-C data to physical distances and describe a Markov chain Monte Carlo (MCMC) approach called MCMC5C to generate a representative sample from the posterior distribution over structures from IF data. Structures produced from parallel MCMC runs on the same dataset demonstrate that our MCMC method mixes quickly and is able to sample from the posterior distribution of structures and find subclasses of structures. Structural properties (base looping, condensation, and local density) were defined and their distribution measured across the ensembles of structures generated. We applied these methods to a biological model of human myelomonocyte cellular differentiation and identified distinct chromatin conformation signatures (CCSs) corresponding to each of the cellular states. We also demonstrate the ability of our method to run on Hi-C data and produce a model of human chromosome 14 at 1Mb resolution that is consistent with previously observed structural properties as measured by 3D-FISH.ConclusionsWe believe that tools like MCMC5C are essential for the reliable analysis of data from the 3C-derived techniques such as 5C and Hi-C. By integrating complex, high-dimensional and noisy datasets into an easy to interpret ensemble of three-dimensional conformations, MCMC5C allows researchers to reliably interpret the result of their assay and contrast conformations under different conditions.Availabilityhttp://Dostielab.biochem.mcgill.ca

Chromatin conformation signatures of cellular differentiation

One of the major genomics challenges is to better understand how correct gene expression is orchestrated. Recent studies have shown how spatial chromatin organization is critical in the regulation of gene expression. Here, we developed a suite of computer programs to identify chromatin conformation signatures with 5C technology http://Dostielab.biochem.mcgill.ca. We identified dynamic HoxA cluster chromatin conformation signatures associated with cellular differentiation. Genome-wide chromatin conformation signature identification might uniquely identify disease-associated states and represent an entirely novel class of human disease biomarkers.

Systematic review and meta-analysis of the effect of North American working hours restrictions on mortality and morbidity in surgical patients

Short duty hours, imposed by the Accreditation Council of Graduate Medical Education (ACGME) regulations, have been claimed to be associated with loss of continuity of care among surgical patients, leading to a potentially increased risk of adverse surgical outcomes. This systematic review and meta‐analysis assessed the strength of associations between duty hour restrictions and morbidity and mortality of various surgical procedures.

Hox in motion: tracking HoxA cluster conformation during differentiation

Three-dimensional genome organization is an important higher order transcription regulation mechanism that can be studied with the chromosome conformation capture techniques. Here, we combined chromatin organization analysis by chromosome conformation capture-carbon copy, computational modeling and epigenomics to achieve the first integrated view, through time, of a connection between chromatin state and its architecture. We used this approach to examine the chromatin dynamics of the HoxA cluster in a human myeloid leukemia cell line at various stages of differentiation. We found that cellular differentiation involves a transient activation of the 5′-end HoxA genes coinciding with a loss of contacts throughout the cluster, and by specific silencing at the 3′-end with H3K27 methylation. The 3D modeling of the data revealed an extensive reorganization of the cluster between the two previously reported topologically associated domains in differentiated cells. Our results support a model whereby silencing by polycomb group proteins and reconfiguration of CTCF interactions at a topologically associated domain boundary participate in changing the HoxA cluster topology, which compartmentalizes the genes following differentiation.

Classifying leukemia types with chromatin conformation data.

BackgroundAlthough genetic or epigenetic alterations have been shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed.ResultsHere, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data.ConclusionsOur study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes.

Acute care surgery in Rwanda: Operative epidemiology and geographic variations in access to care

BACKGROUND Surgical management of emergent, life-threatening diseases is an important public health priority. The objectives of this study were to (1) describe acute care general surgery procedures performed at the largest referral hospital in Rwanda and (2) understand the geographic distribution of disease presentations and referral patterns. METHODS We performed a retrospective review of prospectively collected acute care surgery cases performed at the Centre Hospitalier Universitaire de Kigali (CHUK) in Rwanda between June 1 and December 1, 2011. Using Pearsons χ(2) test and the Fisher exact test, we compared cases originating from within Kigali and transfers from other provinces. Geospatial analyses also were used to further describe transfer patterns. RESULTS During the study period, 2,758 surgical interventions were performed, of which 25.6% (707/2,758) were general surgery operations. Of these, 45.4% (321/707) met the definition of acute care surgery. Only about one-third-32.3% (92/285)-of patients resided within Kigali, whereas about two-thirds-67.7% (193/285)-were transferred from other provinces. Most patients transferred from other provinces were younger than 18 years of age (40.4%; 78/193), and 83.0% (39/47) of patients older than 50 years of age originated from outside of Kigali. Specific operative indications and surgical procedures varied substantially between patients from Kigali and patients transferred from other provinces. CONCLUSION Emergency surgical conditions remain important contributors to the global burden of disease, particularly in low- and middle-income countries. Geographic variations exist in terms of operative diagnoses and procedures, which implies a need for improved access to surgical care at the district level with defined transfer mechanisms to greater-level care facilities when needed.

Abstract 1394: Acute bacterial infection increases lung cancer metastasis via toll-like receptors 2 and 4

INTRODUCTION: Surgery is required for curative treatment of lung cancer but is associated with a high rate of bacterial infections such as pneumonia. There is emerging clinical evidence to suggest that infectious complications may decrease survival after cancer surgery, but the mechanism is unclear. Interactions between bacteria and host cells are mediated by specialized pathogen recognition receptors, of which the Toll-like receptors (TLRs) are best characterized. We and others have shown that cancer cells express TLRs, but their function is largely unknown. The aim of this study is to investigate the mechanisms of bacterial infection-facilitated lung cancer metastasis with a particular focus on the role of TLRs. METHODS: Two lung cancer cell lines (murine H-59 and human A549) were incubated a) directly with heat-inactivated bacteria (S.pneumonia or E.coli) or purified bacterial antigens (lipopolysaccharide or lipoteichoic acid), or b) indirectly with an in vitro pneumonia model using conditioned media from pulmonary bronchial epithelial cells incubated with heat-inactivated bacteria or bacterial antigens. Migratory and metastatic ability of thus treated cells were determined in vitro (adhesion, migration) and in vivo (hepatic intravital microscopy and intra-splenic liver metastasis). A clinically relevant in vivo model of severe infection, cecal ligation and puncture (CLP) or sham control, was employed in some animals. To assess the role of TLRs, we used transgenic TLR4 knockout mice, and small molecule inhibiting molecules or antibodies of TLRs. RESULTS: Direct and indirect (in vitro pneumonia) infection of H-59 and A549 increased adhesion to extracellular matrix components approximately 2-8 fold and 2-4 fold over negative control, respectively. In vitro pneumonia increased in vitro migration of H-59 approximately 2-5 fold. Direct and indirect incubation of H-59 with S.pneumonia and E.coli increased the in vivo adhesion to liver sinusoids 2-4 fold. CLP (in vivo infection model) enhanced the in vivo adhesion of H-59 3-fold and increased number of liver metastases at 2 weeks post-injection approximately 12 fold. These effects were attenuated in TLR4 -/- mice and with TLR2 and TLR4 inhibition. CONCLUSION: Activation of TLR2 or TLR4 on lung cancer cells, either directly by S.pneumonia and E.coli, indirectly though activated bronchial epithelial cells, or via an in vivo infection, increases cancer cell adhesion, migration and metastasis. TLRs thus represent a potential therapeutic target in bacterial infection facilitated cancer metastasis. 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 1394. doi:1538-7445.AM2012-1394

Abstract 5279: LPS-TLR4 binding increases metastatic potential of colorectal cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objective: Infectious complications in colorectal cancer patients undergoing curative resection are associated with cancer recurrence, but the exact mechanism is not well understood. Toll-like receptor 4 (TLR4) is the sole receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen involved in such infectious complication. TLR4 expression has been found on several cancer types. We hypothesize that LPS promotes cancer cell metastatic potential via TLR4 signaling in colorectal cancer cells. Methods: We compared the effects of LPS stimulation on two well-characterized colorectal cancer cell lines: Caco-2 (non-metastatic) and HT-29 (metastatic). Flow cytometry was performed to detect cell surface TLR4 and various integrin expression. Cancer cells were incubated with or without 0.1 µg/mL LPS for 4 hours. In vitro static adhesion assay was used to study the ability of cell adhesion to extracellular matrix proteins (fibronectin, collagen I and collagen IV). Intravital microscopy, a technique used to study microcirculation in vivo, was carried out to observe dynamic attachment of intra-splenic injected cancer cells to C57/BL6 mouse liver sinusoids. Results: Caco-2 cells lack TLR4 receptors and are unresponsive to LPS treatment. In contrast, HT-29 cells were shown to express TLR4 receptors and HT-29 responds to LPS by a 2-fold increase of β1, but not α5, integrin expression in LPS-treated HT-29 cells (Mean Fluorescence Intensity:1041 vs. 458(untreated)). LPS-treated HT-29 cells showed enhanced attachment to fibronectin (1.6-fold; p < 0.01), collagen I (3-fold; p < 0.01) and collagen IV (2-fold; P < 0.01) relative to controls in vitro. Similarly, adherence of HT-29 cells to hepatic sinusoids in vivo was increased by LPS treatment (2 fold; p < 0.05). Conclusion: These data suggest that LPS-TLR4 signaling in colorectal cancer cells increases the metastatic potential. LPS-TLR4 mediated signaling cascade may be a valuable therapeutic target for the prevention of cancer metastasis. 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 5279.