Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan- or oxaliplatin-specific resistance profiles, with non-reciprocal cross-resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance-associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models.

Abstract Tissue inhibitor of metalloproteinases 1 (Timp-1), is one of the four known endogenous inhibitors of matrix metalloproteinases (MMPs); in recent years, Timp-1 has become increasingly recognized as a multifunctional protein that, independently of its MMP inhibitory activity, is able to regulate core cellular processes such as cell proliferation and apoptosis. Consistent with this pro-survival function, Timp-1 expression was shown to be able to protect cancer cells from epirubicin or paclitaxel-mediated cytotoxicity. Consistent with this effect, clinical studies have shown high Timp-1 tumor levels to be predictive of resistance to adjuvant anthracycline-based chemotherapy in metastatic breast cancer patients. In spite of abundant evidence directly involving Timp-1 in regulation of cell growth and apoptosis, the downstream mechanisms of Timp-1–mediated cell signaling underlying these effects, and its biological consequences, have remained unclear. In order to address this issue, we aimed to identify cellular binding partners for Timp-1, which may be able to induce signaling. Therefore, we performed yeast two hybrid screening using a mammary gland cDNA library. We report here the identification of a novel Timp-1 interactor, CD74. CD74, also known as MHC class II invariant chain (li), was mainly thought to function as an MCH class II chaperone promoting the exit of MHC class II molecules from the endoplasmic reticulum (ER). However, a fraction of cellular CD74 has been found to traffic to the plasma membrane where it functions as an accessory-signaling molecule, being quickly recycled back into the endosomal pathway. The interaction between TIMP-1 and CD74 was confirmed by co-immunoprecipitation studies in the triple negative breast cancer cell line MDA-MB-231, and we showed that CD74 is necessary for Timp-1 cellular internalization and Timp-1-mediated activation of Akt signaling. To determine the applicability of our findings to a broader context, we analyzed a breast cancer patient cohort for expression of CD74, CD63 and Timp-1 by immunohistochemistry (IHC) and in situ hybridization (ISH). We found that cancer cells which were negative for TIMP-1 mRNA but positive for Timp-1 protein, indicating an active transport of Timp-1 into the cells. These results raise the possibility that CD74 may be a useful target for effecting Timp-1 mediated chemoresistance. Citation Format: José M Moreira, Mikkel Høeberg, Ulrik Ulrik Lademann, Birgitte Viuff, Lena V Jensen, Jan Stenvang, Sune B Nygård, Maj S Ørum-Madsen, Mette V Vistesen, Anja T Fuglsang, Siqi Liu, Nils Brünner. Identification and characterization of a new TIMP-1 binding protein [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-08.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.

Background: The influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically and the infection dynamics compared to an "avian-like" H1N1 virus by an experimental infection study.Methods: Sequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an "avian-like" H1N1 virus, respectively, followed by inoculation with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR.Results: The phylogenetic analysis revealed that the reassorted H1N2 virus contained a European "avian-like" H1-gene and a European "swine-like" N2-gene, thus being genetically distinct from most H1N2 viruses circulating in Europe, but similar to viruses reported in 2009/2010 in Sweden and Italy. Sequence analyses of the internal genes revealed that the reassortment probably arose between circulating Danish "avian-like" H1N1 and H3N2 SIVs. Infected pigs developed cross-reactive antibodies, and increased levels of acute phase proteins after inoculations. Pigs inoculated with H1N2 exhibited nasal virus excretion for seven days, peaking day 1 after inoculation two days earlier than H1N1 infected pigs and at a six times higher level. The difference, however, was not statistically significant. Pigs euthanized on day 4 after inoculation, had a high virus load in all lung lobes. After the second inoculation, the nasal virus excretion was minimal. There were no clinical sign except elevated body temperature under the experimental conditions.Conclusions: The "avian-like" H1N2 subtype, which has been established in the Danish pig population at least since 2003, is a reassortant between circulating swine "avian-like" H1N1 and H3N2. The Danish H1N2 has an "avian-like" H1 and differs from most other reported H1N2 viruses in Europe and North America/Asia, which have H1-genes of human or "classical-swine" origin, respectively. The variant seems, however, also to be circulating in countries like Sweden and Italy. The infection dynamics of the reassorted "avian-like" H1N2 is similar to the older "avian-like" H1N1 subtype.

Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus and the infections with human respiratory syncytial virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared by neutrophils whereas apoptosis was an important way of clearance of BRSV-infected epithelial cells. Neighboring cells, which probably were epithelial cells, phagocytized the BRSV-infected apoptotic cells. The number of both CD4(+) and CD8+ T cells increased during the course of infection, but the T cells were not found between the epithelial cells of the bronchi up until apoptosis was no longer detected, thus in the bronchi there was no indication of direct contact-dependent T-cell-mediated cytotoxicity in the primary infection.