Article

Mutation-specific effects of NRAS oncogenes in colorectal cancer cells

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Abstract

In colorectal cancer (CRC), the prevalence of NRAS mutations (5–9%) is inferior to that of KRAS mutations (40–50%). NRAS mutations feature lately during tumour progression and drive resistance to anti-EGFR therapy in KRAS wild-type tumours. To elucidate specific functions of NRAS mutations in CRC, we expressed doxycycline-inducible G12D and Q61K mutations in the CRC cell line Caco-2. A focused phospho-proteome analysis based on the Bio-Plex platform, which interrogated the activity of MAPK, PI3K, mTOR, STAT, p38, JNK and ATF2, did not reveal significant differences between Caco-2 cells expressing NRASG12D, NRASQ61K and KRASG12V. However, phenotypic read-outs were different. The NRAS Q61K mutation promoted anchorage-independent proliferation and tumorigenicity, similar to features driven by canonical KRAS mutations. In contrast, expression of NRASG12D resulted in reduced proliferation and apoptosis. At the transcriptome level, we saw upregulation of cytokines and chemokines. IL1A, IL11, CXCL8 (IL-8) and CCL20 exhibited enhanced secretion into the culture medium. In addition, RNA sequencing results indicated activation of the IL1-, JAK/STAT-, NFκB- and TNFα signalling pathways. These results form the basis for an NRASG12D-driven inflammatory phenotype in CRC.

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Ras controls a multitude of cellular signaling processes, including cell proliferation, differentiation, and apoptosis. Deregulation of Ras cycling often promotes tumorigenesis and various other developmental disorders, termed RASopothies. Although the structure of Ras has been known for many decades, it is still one of the most highly sought-after drug targets today, and is often referred to as "undruggable." At the center of this paradoxical protein is a lack of understanding of fundamental differences in the G domains between the highly similar Ras isoforms and common oncogenic mutations, despite the immense wealth of knowledge accumulated about this protein to date. A shift in the field during the past few years toward a high-resolution understanding of the structure confirms the hypothesis that each isoform and oncogenic mutation must be considered individually, and that not all Ras mutations are created equal. For the first time in Ras history, we have the ability to directly compare the structures of each wild-type isoform to construct a "base-line" understanding, which can then be used as a springboard for analyzing the effects of oncogenic mutations on the structure-function relationship in Ras. This is a fundamental and large step toward the goal of developing personalized therapies for patients with Ras-driven cancers and diseases.
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Inflammation occurs after disruption of tissue homeostasis by cell stress, injury or infection and ultimately involves the recruitment and retention of cells of hematopoietic origin, which arrive at the affected sites to resolve damage and initiate repair. Interleukin 1α (IL-1α) and IL-1β are equally potent inflammatory cytokines that activate the inflammatory process, and their deregulated signaling causes devastating diseases manifested by severe acute or chronic inflammation. Although much attention has been given to understanding the biogenesis of IL-1β, the biogenesis of IL-1α and its distinctive role in the inflammatory process remain poorly defined. In this review we examine key aspects of IL-1α biology and regulation and discuss its emerging importance in the initiation and maintenance of inflammation that underlie the pathology of many human diseases.
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Purpose: This review aimed to take stock of the current status of research on damage-associated molecular pattern (DAMP) protein. We discuss the Janus-faced role of DAMP molecules in inflammation, cancer, and tissue repair. The high-mobility group box (HMGB)-1 and adenosine triphosphate proteins are well-known DAMP molecules and have been primarily associated with inflammation. However, as we shall see, recent data have linked these molecules to tissue repair. HMGB1 is associated with cancer-related inflammation. It activates nuclear factor kB, which is involved in cancer regulation via its receptor for advanced glycation end-products (RAGE), Toll-like receptors 2 and 4. Proinflammatory activity and tissue repair may lead to pharmacologic intervention, by blocking DAMP RAGE and Toll like receptor 2 and 4 role in inflammation and by increasing their concentration in tissue repair, respectively. Methods: We conducted a MEDLINE search for articles pertaining to the various issues related to DAMP, and we discuss the most relevant articles especially (ie, not only those published in journals with a higher impact factor). Findings: A cluster of remarkable articles on DAMP have appeared in the literature in recent years. Regarding inflammation, several strategies have been proposed to target HMGB1, from antibodies to recombinant box A, which interacts with RAGE, competing with the full molecule. In tissue repair, it was reported that the overexpression of HMGB1 or the administration of exogenous HMGB1 significantly increased the number of vessels and promoted recovery in skin-wound, ischemic injury. Implications: Due to the bivalent nature of DAMP, it is often difficult to explain the relative role of DAMP in inflammation versus its role in tissue repair. However, this point is crucial as DAMP-related treatments move into clinical practice.
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The idea of a potential link between cancer and inflammation was first proposed by R. Virchow in the nineteenth century. However, clear evidence regarding a key role of inflammation in oncogenesis appeared only during the last decade. Now the tumor microenvironment is commonly considered as an obligatory and significant component of almost all types of cancer, and the cells infiltrating such microenvironment are a source of inflammatory cytokines. Such cytokines play a key role in regulating inflammation during both normal immune response and developing cancer. In this review, we explore the role of two inflammatory cytokines interleukin 1 and interleukin 6 in cancer development. These cytokines have pleiotropic effects on various cell types in the tumor microenvironment, particularly being able to regulate pro-oncogenic transcription factors NF-κB and STAT3. For this reason, such cytokines influence key parameters of oncogenesis, increasing cell resistance to apoptosis, proliferation of cancer cells, angiogenesis, invasion and malignancy as well as the ability of tumor cells to respond to anticancer therapy. Here we summarize novel experimental data regarding mechanisms underlying the interaction between chronic inflammation and malignant neoplasms.
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The human epithelial cell line Caco–2 has been widely used as a model of the intestinal epithelial barrier. The Caco–2 cell line is originally derived from a colon carcinoma. However, one of its most advantageous properties is its ability to spontaneously differentiate into a monolayer of cells with many properties typical of absorptive enterocytes with brush border layer as found in the small intestine. The Caco-2 cell line is heterogeneous and contains cells with slightly different properties. Thus, cultivation conditions can be expected to select for the growth of subpopulations of cells resulting in a cellular model system with properties that may differ from the original cell line. Accordingly, results obtained under similar experimental conditions in different laboratories may not be directly comparable. Due to this, a variety of cloned Caco–2 cell lines has been established, and described in the literature. This chapter will however, focus on describing how to handle and cultivate the original Caco–2 cell line as obtained from cell culture collections like American Type Culture Collection and the European Collection of Cell Cultures. Detailed protocols for handling the Caco-2 cells in the laboratory are provided. Furthermore, in Chap.9 general protocols for measuring barrier function by transepithelial resistance (TEER), and monolayer integrity by Lucifer Yellow flux are described. Proper testing of the cell monolayer is absolutely critical in exploiting Caco–2 cells to measure interaction, uptake and cellular transport of drugs and food components.
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Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC-released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α was released by injured colonocytes, thereby exacerbating experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α-positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1β. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and induced de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α-mediated IEC-fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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Neuroblastoma rat sarcoma (RAS) viral oncogene homolog (NRAS), a small GTPase, is one of the most thoroughly studied oncogenes that controls cell growth, differentiation, and survival by facilitating signal transduction. Here, we identify four novel naturally occurring NRAS isoforms (isoforms 2-5) in addition to the canonical isoform (isoform 1). Expression analyses performed on a panel of several different human malignancies and matching normal tissue revealed distinct isoform expression patterns. Two of the novel isoforms were found in the nucleus and cytoplasm, whereas the others were exclusively cytoplasmic. The isoforms varied in their binding affinities to known downstream targets and differentially regulated the RAS signaling pathway. Strikingly, forced expression of isoform 5, which encodes only a 20-aa peptide, led to increased cell proliferation and to transformation by activation of known NRAS targets. These discoveries open new avenues in the study of NRAS.
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The presence of sucrase-isomaltase (SI), a glycoprotein hydrolase normally restricted to the brush border membrane of the enterocytes of the small intestine, was investigated in tumours which developed in nude mice inoculated with six human colon carcinoma cell lines (HT-29, Caco-2, HRT-18, HCT-8R, SW-480, and CO-115). Foetal and normal adult human small intestines and colons were used as controls. SI was studied by (I) immunofluores-cence with rabbit antibodies raised against purified human small intestine SI; (2) polyacrylamide gel elec-trophoresis and immunoblotting; and (3) determination of the enzyme activity. SI was antigenically present, and enzymatically active, in all the tumours derived from Caco-2 and HT-29 cells. The presence of the enzyme was associated with that of typical brush borders at transmission electron microscopy examination. SI was absent from the tumours developed with the other four cell lines, as well as from the normal adult colon mucosa. SI was also present and active in the colons of mid-gestation foetuses, ranging in ages between 20 and 28 weeks; it was absent from the colons of late-gestation foetuses. The presence of SI in tumours derived from two cell lines suggests that this enzyme is a marker, so far unsuspected, of certain human colon cancers, and that the differentiation pattern of these particular cancers closely resembles that of the foetal colon.
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Chemokines (chemo-attractant cytokines) are a group of small proteins that act together with their cell surface receptors, in development, normal physiology and immune responses, to direct cells to specific locations throughout the body. Cancer cells acquire the ability to subvert the chemokine system, such that these molecules and their receptors become important regulators of cell movement into and out of the tumour microenvironment and major players in cancer biology. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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The mediators and cellular effectors of inflammation are important constituents of the local environment of tumours. In some types of cancer, inflammatory conditions are present before a malignant change occurs. Conversely, in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumours. Regardless of its origin, 'smouldering' inflammation in the tumour microenvironment has many tumour-promoting effects. It aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to hormones and chemotherapeutic agents. The molecular pathways of this cancer-related inflammation are now being unravelled, resulting in the identification of new target molecules that could lead to improved diagnosis and treatment.
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Numerous cell lines derived from human tumors are not HeLa contaminants. Of 192 lines established in this or other laboratories, 169 lines were found to be G6PD type B. Twenty-three lines were type A as HeLa; three of these were of Negroid origin. There is reasonable doubt that the remaining 20 lines will all be shown to be confounded with HeLa.
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Subcloning of interleukin 3 (IL-3)-dependent PB-3c mastocyte cells revealed two populations, of which only one is sensitive to oncogenic transformation by v-H-ras. The corresponding tumors produce IL-3 and grow in vitro in the absence of exogenous IL-3 [Nair, A.P.K., Diamantis, I.D., Conscience, J.F., Kindler, V., Hofer, P. & Moroni, Ch. (1989). Mol. Cell. Biol., 9, 1183-1190]. In the present investigation, IL-3 gene regulation was compared in ras transformable (rT) and ras nontransformable (rNT) lines. We report that upon expression of v-H-ras rT clones but not rNT clones express low levels of IL-3 mRNA as detected by reverse polymerase chain reaction. Treatment with ionomycin, a calcium ionophore, induced high levels of IL-3 expression only in ras-expressing rT clones. Somatic cell fusion between the rNT clone 20 and the IL-3-expressing mastocytoma line V2D1 led to down-regulation of IL-3 expression and to the requirement for exogenous IL-3 for in vitro growth and tumor suppression. In contrast, rT clone 15 lacked tumor-suppressor activity and failed to down-regulate IL-3 expression in somatic hybrids which grew in vitro without added IL-3. Our results indicate that IL-3 gene expression is a critical determinant for the generation of v-H-ras-induced mast cell tumors and show that disturbances in IL-3 gene regulation can be detected already at the premalignant level in v-H-ras transformation-sensitive cells.
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A complex network of chemokines and their receptors influences the development of primary tumours and metastases. New information about the biological role of chemokines in these processes is providing insights into host–tumour interactions, such as the role of the leukocyte infiltrate, and into the mechanisms that determine the metastatic potential and site-specific spread of cancer cells. Chemokine-receptor antagonists are showing promise in animal models of inflammation and autoimmune disease. Could manipulating the local chemokine network have therapeutic benefits in malignant disease?
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Colorectal cancer arises after a series of mutational events in the colon epithelia and is often used as a model of the multistep progression of tumorigenesis. Mutations in Ki-Ras have been detected in some 50% of cases and are thought to occur at an early stage. Almost never do mutations arise in the loci of other Ras isoforms (Ha- and N-), leading to the assumption that Ki-Ras plays a unique role in tumorigenesis. In order to examine the distinctive function that Ki-Ras plays in cancer development in the colon, we introduced constitutively active mutant Ki- and Ha-Ras genes into an intermediate-stage colon adenoma cell line (Caco-2). We found that mutant active Ha-RasV12 was more efficient at transforming these colon epithelial cells as assessed by anchorage-independent growth, tumor formation in SCID mice and the development of mesenchymal morphology compared to transformation by Ki-RasV12. We conducted microarray analysis in an attempt to reveal the genes whose aberrant expression is a direct result of overexpression of either Ki-RasV12 or Ha-RasV12. We used Clontech's Atlas cancer cDNA (588 genes) and RZPD's Onco Set 1 (1,544 genes) arrays. We identified fewer genes that were commonly regulated than were differentially expressed between Ki- and Ha-RasV12 isoforms. Specifically, we found that Ki-RasV12 regulated genes involved in cytokine signaling, cell adhesion and colon development, whereas Ha-RasV12 mainly regulated genes involved in controlling cell morphology, correlating to an epithelial-mesenchymal transition only observed in these cells. Our results demonstrate how 2 Ras isoforms regulate disparate biologic processes, revealing a number of genes whose deregulated expression may influence colon carcinogenesis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).
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Cytokines such as IL-1 and TNF are primarily pro-inflammatory. The inflammation induced by these cytokines is reflected in the type of genes they induce. In the pathogenesis of carcinogenesis as well as tumor growth and spread, cytokines such as IL-1 and TNF induce chemokines that attract neutrophils. Neutrophils are key players in the production of reactive oxygen species and carcinogenesis. Another aspect of pro-inflammatory cytokines is the induction of adhesion molecules and metalloproteinases, both of which provide mechanisms for tumor invasion. Blocking cytokines, however, will reduce tumor growth and spread if administered at sufficient concentrations and will require parenteral therapy. However, blocking cytokines will not kill tumor cells nor prevent carcinogenesis. Blocking cytokines is best as an adjunct therapy together with tumorocidal drugs.
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Tumor-associated macrophages (TAM) are key regulators of the link between inflammation and cancer. In the tumor microenvironment neoplastic cells shape the differentiation and functional orientation of TAM which, in turn, express several protumoral functions, including secretion of growth factors and matrix-proteases, promotion of angiogenesis and suppression of adaptive immunity. This review analyzes our current knowledge of TAM and their involvement in tumor development and progression. The interplay between TAM and neoplastic cells represents a promising target of future therapeutic approaches.
The chemokine system and cancer
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Cancer and the chemokine network
  • Balkwill