Maria Kalipciyan

Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling

Resistance to anticancer drugs such as the widely used antimetabolite 5‐fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low‐, intermediate‐ and high‐resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM‐2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium‐binding protein family were suppressed. Acquisition of the intermediate‐resistance phenotype included upregulation of the well‐known drug resistance gene ABCC6 (ATP‐binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time‐depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high‐resistance phenotype seems to be less demanding.

KRAS mutations in historical tumour specimens of the Viennese Museum of pathological anatomy.

Sir: The Viennese Museum of Pathological Anatomy displays worldwide one of the largest collection of specimens. It comprises more than 50 000 exhibits, including approximately 27 000 formalin-fixed samples of a variety of diseased organs. This collection comprises primarily extraordinary and unique objects, which were used mainly for presentation and exhibitions. While palaeopathological studies on ancient material are common, only few studies dealt with historical specimens from the 19th century. Although ancient DNA samples of tumours have been studied, historical tumour exhibits, to the best of our knowledge, have not yet been investigated. We thus focused upon tumours of the 19th and early 20th century in order to look for relevant mutations that developed under different environmental conditions. Thirty-one tumour specimens were selected, ranging in age from 79 to 194 years. Five slices (5 lm) of paraffin-embedded tissue were treated with ethanol and incubated overnight to digest proteins. Finally, DNA was precipitated, washed, dissolved and stored at )20 C until polymerase chain reaction (PCR) analysis. As photometric analysis was not feasible due to the small yield of isolated gDNA, a PCR method has been Figure 1. Breast carcinoma of the 19th century grossly and histologically.

Exosomal microRNA transfer varies with specific microRNAs functional in colorectal cancer and cellular differentiation.

Exosomes are membranous microparticles (40 – 100 nm diameters) of endocytic origin that are released by a variety of cell types into the extracellular space [1]. These particles consist of a lipid bilayer membrane surrounding a small cytosol and contain various molecular constituents of their cell of origin, including proteins and nucleic acid material (all types of RNAs) [2]. MicroRNAs (miR), which are incorporated into exosomes as well, are short (19 – 24 nt) noncoding RNAs that play important roles in posttranscriptional gene silencing of target messenger RNAs [3]. It has been demonstrated that exosomes are ”bioactive vesicles” as they promote intercellular communication and immunoregulatory processes by shuttling their containing molecules from one cell to another [4, 5]. In cancerous tissue, the expression of microRNAs and their exosomal transmission is often deregulated [6]. The aim of this study was to evaluate the incorporation of five functional microRNAs relevant in colorectal cancerous tissue due to their often dysregulated expression into exosomes (miR-10b-5p, miR-21-5p, miR141-3p, miR-200c-3p and miR-375). Furthermore, we provide insight into exosomal microRNA transport in the context of cellular differentiation. Materials and methods

Abstract A12: Differential micro-RNA expression in a multistage 5-fluorouracil resistant colon cancer model

Purpose: Among small non-coding RNAs, micro-RNAs have demonstrated a variety of essential functions under both physiological and pathological conditions such as cell cycle progression, oncogenic transformation and apoptosis. Thus, we hypothesized that micro-RNAs may play a major role in cellular stress adaptation. Methods: To attain this aim, we evaluated a multi-stage colon cancer system with increasing degrees of resistance to the antimetabolite 5-fluorouracil by i) differential expression profiling (micro-RNA arrays) and ii) subsequent validation of the candidate RNAs by quantitative PCR. Results: Among 15 differential expressed micro-RNAs in our model system, 9 were confirmed by qPCR and selected for further investigations. Noteworthy, for every level of drug resistance (low, intermediate, and high) specific micro-RNAs were identified indicating an individual response to escalating cytotoxic stress at the molecular level. With regard to micro-RNA functions, our findings point at an involvement - if not a regulatory function - of the recognized micro-RNAs in the following processes (data from the literature): doxorubicin resistance (miR-130a), survival signaling, DNA-oriented stress, and oncogenic properties (all miR-19), epithelial-mesenchymal transition (miR-141 and miR-200), as well as invasion and metastasis (miR-10b). Some of these features are currently under investigation by transfection of the corresponding micro-RNA precursors or antagomirs in the chemoresistant model in order to assess the contribution of the identified micro-RNAs to the resistance phenotype. Conclusions: Previous work from our team has demonstrated the orchestrated response of cancer cells to xenobiotics at the molecular level by global gene expression profiling. The investigated subclones, derived from the metastatic colon cancer cell line CCL 227, were characterized by stage dependent mRNA regulations affecting vital cellular functions such as cytoskeleton, cell-cell communication, signal transduction, cell cycle control and apoptosis. Remarkably, different degrees of chemoresistance addressed different cellular mechanisms, thus providing to the cancer cell the necessary flexibility to adapt to different levels of stress. Likewise, micro-RNAs seem to follow a similar behavior with different micro-RNA patterns reflecting the development of resistance to 5-fluorouracil. As micro-RNAs are very likely to play a decisive role in the cellular response to therapeutic stress, their regulation might offer novel starting points to understand and to oppose drug resistance. This work has been supported by the “Initiative Krebsforschung”, Austria. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A12