The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer

Biochimica et Biophysica Acta 1795 (2009) 37–52

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a c a n

Review

The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer Jaime Acquaviva a, Ricky Wong b, Al Charest a,b,c,⁎ a b c

Molecular Oncology Research Institute, Tufts University School of Medicine, Tufts Medical Center, Boston, Massachusetts, USA Department of Neurosurgery, Tufts University School of Medicine, Tufts Medical Center, Boston, Massachusetts, USA Department of Biology and The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

a r t i c l e

i n f o

Article history: Received 1 May 2008 Accepted 21 July 2008 Available online 3 August 2008 Keywords: Cancer c-ROS Glioblastoma Signal Transduction Receptor Kinase

a b s t r a c t The proto-oncogene receptor tyrosine kinase ROS was originally discovered through the identification of oncogenic variants isolated from tumors. These discoveries spearheaded a body of work aimed at elucidating the function of this evolutionarily conserved receptor in development and cancer. Through genetic and biochemical approaches, progress in the characterization of ROS points to distinctive roles in the program of epithelial cell differentiation during the development of a variety of organs. Although substantial, these advances remain hampered by the absence of an identified ligand, making ROS one of the last two remaining orphan receptor tyrosine kinases. Recent studies on the oncogenic activation of ROS as a result of different chromosomal rearrangements found in brain and lung cancers have shed light on the molecular mechanisms underlying ROS transforming activities. ROS and its oncogenic variants therefore constitute clinically relevant targets for cancer therapeutic intervention. This review highlights the various roles that this receptor plays in multiple system networks in normalcy and disease and points to future directions towards the elucidation of ROS function in the context of ligand identification, signaling pathways and clinical applications. © 2008 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An evolutionarily conserved receptor . . . . . . . . . . . . . . . . . . . . . c-Ros expression in development and adults: Avian and rodent species . . . . . 4.1. c-Ros expression in the kidney and intestinal tract . . . . . . . . . . . . 4.2. c-Ros expression in the lung . . . . . . . . . . . . . . . . . . . . . . 4.3. c-Ros expression in reproductive tissues . . . . . . . . . . . . . . . . c-ROS expression in humans . . . . . . . . . . . . . . . . . . . . . . . . . c-ROS Expression in disease . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Cancers of the central nervous system . . . . . . . . . . . . . . . . . 6.2. Lung cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Breast, stomach, liver, kidney and colon cancers . . . . . . . . . . . . . 6.4. Heart disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenic ROS fusion kinases . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. V-ROS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. FIG-ROS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. SLC34A2-ROS and CD74-ROS . . . . . . . . . . . . . . . . . . . . . . Epithelial differentiation; insights into the phenotype of ROS-null mice . . . . . ROS elicits signal transduction programs of proliferative growth and cell survival 9.1. SH2 Domain containing tyrosine phosphatases SHP-1 and SHP-2 . . . . . 9.2. MAP kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. The PI3K and Akt pathway . . . . . . . . . . . . . . . . . . . . . . . 9.4. STAT3 Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5. VAV3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Tufts University School of Medicine, 800 Washington Street, Tufts Medical Center Box 5609, Boston, Massachusetts 02111 USA. Tel.: +1 617 636 8876; fax: +1 617 636 5277. E-mail address: [email protected] (A. Charest). 0304-419X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2008.07.006

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10. Ligand(s) . . 11. Conclusion . Acknowledgments References . . . .

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1. Introduction Receptor tyrosine kinases (RTKs) are important regulators of cellular signal transduction pathways that play crucial roles in development and cancer by regulating cellular proliferation, differentiation, migration and death. Not surprisingly, perturbations in RTK signaling through various genetic alterations result in deregulated kinase activity and ensuing malignant transformation. In humans, there are 58 RTKs falling into 20 distinct architecturally defined families (for a review see [1]). Identifying the physiological regulations of RTKs is necessary in order to understand the mechanisms causing their oncogenic activation and to devise counteracting approaches as means of therapeutic interventions. Typically, signaling by RTKs is initiated by ligand-induced receptor oligomerization, which structurally allows for autophosphorylation of the receptor subunit. Recent mechanistic insights based on X-ray crystallography studies of various RTKs demonstrate a common theme of kinase activation through structural changes (for a review see [2]). This rearrangement further activates the catalytic activity of the receptors and produces phosphorylated tyrosine residues which are now the targets of specific and high affinity binding of cytoplasmic signaling proteins containing Src homology-2 (SH2) and protein tyrosine-binding (PTB)

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domains. Creation of such phospho-specific docking sites result in the amplification and dissemination of an extracellular-initiated signal into intracellular information. These tightly regulated signals integrate into a highly orchestrated cellular machinery capable of discerning the most minute of environmental changes. Deregulation of phosphotyrosine-mediated signaling events is unfortunately very common in human cancers. From a clinical standpoint, understanding RTKs activation and pursuing the development of specific inhibitors hold great promise towards the discovery of novel anti-cancer therapeutic agents. 2. Discovery of ROS ROS was discovered more than 20 years ago as the oncogene product of the avian sarcoma RNA tumor virus UR2 (University of Rochester tumor virus 2- originally by R.E. Luginbuhl in 1963 at the University of Connecticut Poultry Diagnostic Lab) [3–5]. In the early 1980s, Balduzzi and colleagues observed that this highly invasive fibrosarcoma-derived virus was efficient at transforming chicken embryo fibroblasts (CEFs) [4,6–9] and chicken embryo neuroretinal cells [10]. These observations were later followed by deciphering the amino acid sequence coded from the onc portion of

Fig. 1. ROS is a evolutionarily conserved receptor tyrosine kinase. (A) Schematic representation ROS and its invertebrate homologues ROL-3 and SEV. The figure is drawn to scale in relation to the position of the FN-III-like repeats (blue boxes), the transmembrane region and the kinase domain (green rectangle). The length of each receptor (number of amino acids) is also indicated. (B) Alignment of the kinase domain sequences of ROS, ROL-3 and SEV demonstrating that they are highly similar to each other. Shown is a matrix of percent identity between each pair of kinase domain as calculated by the Clustal W method.

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UR2 (a 68 kDa polypeptide named p68v-ros) which demonstrated a significant homology to those of analogous regions from other RNA tumor viruses (src, yes, fps, fms, erb B, fgr, abl) and that it similarly coded for a protein with tyrosine kinase activity [6,8,11,12]. Concomitantly, Wigler's group identified an additional oncogenic form of ROS during human genomic DNA transfer experiments in NIH-3T3 cells designed to uncover novel transforming genes [13]. Recovery of tumor-inducing genomic DNA uncovered the mcf3 oncogene, later shown to be a truncated c-ROS product similar in structure to the avian UR2 viral p68v-ros [14]. Sequence comparison of these oncogenic forms of ROS to its then recently available fulllength sequence led to the suggestion that loss of the extracellular domain may result in the activation of this proto-oncogene [14–20]. In fact, like many RNA tumor viruses, the oncogenic potential of p68v-ros resides in the fusion of the viral genome-coded structural p19gag protein to the intracellular kinase domain region of avian cRos and subsequent activation of tyrosine kinase activity [21–24]. To this date, the identity of the sequence upstream to c-ROS in the mcf3 oncogene locus remains unknown. Recently, three additional naturally occurring oncogenic versions of ROS have been reported. First, an interstitial micro deletion on chromosome 6q21 which results in a fusion event between a novel gene called FIG (fused in glioblastoma) (also known as GOPC [25], PIST [26] and CAL [27]) and sequences coding for the intracellular portion of c-ROS was uncovered in human glioblastoma cell lines [28–30]. We have demonstrated that spatio-temporally controlled expression of the FIG-ROS oncogene in the CNS of genetically engineered adult mice results in the formation of glioblastoma multiforme tumors [31]. More recently, two inter-chromosomal translocations resulting in the fusion of the SLC34A2 and CD74 genes to the extracellular juxtamembrane portion of c-ROS have been reported in non-small cell lung cancer cell lines and patient tumors [32]. In two of these cases, expression of such deregulated, ligand independent RTK ROS, has been shown to be a potent oncogenic event. 3. An evolutionarily conserved receptor The Ros receptor tyrosine kinase gene is conserved among Caenorhabditis elegans, Drosophila melanogaster, and vertebrate species (avian and mammalian). In all species, the molecular architecture of the c-ROS gene product consists of an extracellular or ectodomain, a hydrophobic stretch corresponding to a single pass transmembrane spanning region, an intracellular portion containing the tyrosine kinase domain and a carboxyl terminal tail [20,33,34] (see Fig. 1). This structural organization is similar to those of other type I growth and differentiation factor receptors (e.g. EGFR, INSR, FGFR). What impart uniqueness to ROS are its ectodomain composition and the remote similarity of its catalytic domain sequence when compared to other tyrosine kinase domains. Phylogenic relationship analysis of the kinase domains of all 58 known human RTKs demonstrates that ROS is a distinct receptor which is distantly related to the ALK/LTK and INSR families [35]. Perhaps the most striking feature of ROS is its extracellular domain structure. It is composed of a tandem of six repeat motifs with high sequence homology to fibronectin. Fibronectin is an extracellular matrix and plasma protein that plays a critical role in cell adhesion. It contains three kinds of repeats: type I-III [36]. Amino acid sequences analogous to those of the third fibronectin repeat (FN-III-like repeat) are present in a variety of cell surface and extracellular matrix (ECM) proteins from invertebrates to vertebrates. The presence of multiple fibronectin type III-like repeats distributed throughout the ectodomain of ROS is therefore highly reminiscent of many cell adhesion molecules (CAMs). However, unlike most CAMs, ROS also contains an intracellular domain possessing kinase activity. This combination makes ROS unusual in its potential to directly translate adhesion engagement to intracellular signaling pathways.

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Among other members of the RTK family harboring FN-III-like repeats are members of the insulin receptor family (INSR, IGF-1R and IRR), Axl family (MER and TYRO3), TIE family (TEK) and the ephrin receptor (EPH) family. In these receptors, data pertaining to functional attributes of their FN-III-like repeats is scarce. For the INSR and IGF-1R, it appears that the C-terminal portion of the first FN-III-like repeat is involved in mediating a structural requirement for insulin binding [37,38]. For the EPHA3 receptor, binding of the EphrinA5 ligand is mediated through the membrane proximal FN-III-like repeat [39]. To this date, no role for FN-III-like repeats from any of the other RTKs has been reported. Given the importance of the function that these receptors have in many biological systems and in various diseases, elucidating FN-III-like repeats structure–function relationships seems to be a legitimate area to focus research on. Similar to many CAMs, the extracellular domain of ROS was found to contain multiple potential sites of N-linked glycosylation and shown to be glycosylated [20,34,40,41]. These structural features suggest that perhaps ROS has the unique ability to directly couple extracellular adhesion mediated events to tyrosine phosphorylationbased intracellular signaling pathways. Although very little is known regarding the function of the extracellular domain of ROS in any physiological processes, clues to ROS ligand identity and ensuing functions in mammalian systems may be obtained from its nonvertebrate orthologues, the C. elegans ROL-3 and the D. melanogaster SEVENLESS receptors. In C. elegans, the ROS ortholog ROL-3, was originally identified in a screening effort to isolate and analyze chemically induced mutants that appear to have alterations in cuticle structure [42]. The nematode cuticle or exoskeleton is an impervious barrier between the animal and its environment that is essential for the maintenance of body morphology. It is a highly structured extracellular matrix (ECM) that is composed predominantly of cross-linked collagens, insoluble proteins termed cuticlins, associated glycoproteins and lipids (for a review see [43]. During the synthesis of the cuticle, these ECM components are secreted from the apical epithelial membranes of the hypodermis and then polymerize only to remain in intimate contact with the outer surface of these membranes. The rol-3(e754) mutation is a member of a general class of mutations affecting gross morphology through disruption of the nematode cuticle. It was found that worms homozygous for rol-3 (e754) display abnormally left-hand twisted cuticles, body musculature, gut and ventral nerve cords, as well as an aberrant left-handed rotation during locomotion [44]. Barbazuk et al. further generated recessive alleles of rol-3 alleles including a temperature sensitive rol-3 (s1040ts) allele lethal at high temperature but viable albeit weak at low temperature [45]. The identity of each of these allelic mutants of Rol-3 at the molecular level still remains undetermined. Experiments aimed at solving ROL-3′s function in cuticle structure genesis and maintenance led to the delineation of ROL-3 expression patterns. Using a transgene consisting of the Rol-3 promoter driving the expression of eGFP [46], it was found that during embryo and larval stages, the Rol-3 promoter is active in the body wall muscle and the epidermal layer of the hypodermis. In the adult, the promoter is active in the body wall muscle, the vulval muscle of the reproductive system and in unidentified head neuron of the nervous system. These experiments imply that ROL-3 expression and presumably, signaling events are necessary for proper developmentally regulated cues that give rise to the morphology of the animal. Genetic screens for suppressor phenotypes of rol-3(s1040ts) mutation generated two loci; suppressor of roller lethal srl-I and srl2. The molecular identity of these two loci remains to be determined. Interestingly, srl-I localizes to linkage group (LG) II, the same LG where the ptp-2 gene, the C. elegans orthologue of SHP-2, a known direct downstream effector of ROS, is found. Deciphering structure–function relationship of this ROS analog from these rol-3 mutants would certainly further our understanding of ROS regulation and function in

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C. elegans development and could shed light on mammalian ROS functions otherwise unnoticed. Similar to ROL-3, ROS and SEVENLESS exhibit an unusually high degree of gross structural and sequence similarities (Fig. 1) [20,33,34]. During the development of the compound eye of drosophila, differentiation of the various cell types in each ommatidium is controlled by at least two RTKs, the drosophila EGF receptor (DER) and the drosophila SEVENLESS receptor (SEV) [47-49]. DER is known to control the differentiation of most cells in the developing eye and to provide additional cues in proliferation and cell survival controls [5055]. SEV however, is only required for specification of the R7 photoreceptor cell even though it is dynamically expressed in subpopulations of other ommatidial cells [56]. A loss of function mutation in the sev gene result in the failure of the R7 precursor cells to fully differentiate into photoreceptor cells. Large-scale genetic screens conducted in this system unraveled many steps in the SEV signaling cascade. Like many RTKs, activated SEV transduces signals through the small GTPase RAS1 through the activation of the RAS guanine exchange factor protein Son of Sevenless (SOS). Supporting and surrounding this RTK-RAS1 pathway are many additional signaling molecules such as the tyrosine phosphatase CORKSCREW, Downstream of Receptor Kinases (DRK) and daughter of sevenless (DOS), invertebrate homologues of the signaling proteins SHP-2, GRB2 and GAB-1/IRS1 respectively. From these studies emerged a complex system of protein tyrosine kinases and phosphatases acting upon various enzymes and scaffolding proteins designed to transduce a SEV signal into changes in gene expression which leads to neuronal differentiation (for a review see [57]). The exclusive specificity of SEV signaling to the R7 cells was molecularly uncovered through genetic studies which have revealed that the ligand for SEV is a 7 transmembrane G-protein coupled receptor (GPCR) which is located on the surface of the adjacent R8 cells. This protein, termed bride of sevenless (BOSS) represents an unusual ligand for an RTK. Positional proximity of the already differentiated R8 to the non committed R7 cells result in the activation of SEV in the R7 precursor population by binding to the BOSS protein located on the surface of R8 cells. The ensuing activation of SEV result in the differentiation of the R7 cells to their final state of pho`toreceptor [58,59]. In the absence of BOSS, the R7 precursor cells fail to differentiate into photoreceptors but rather develop into a non-neuronal cone cell [56,60]. A major question remains to be addressed. For example, why is it that R7 cells need two RTKs to accomplish differentiation when other photoreceptor cells can perform with only DER signaling? In this system, it appears that SEV activity act in conjunction with another RTK to drive a very specific cell type into a differentiation path. Despite major inroads into the deciphering of SEV signaling, much remains to be clarified in this system. No doubt that uncovering additional SEV-driven biological processes will help elucidate ROS's function in vertebrate systems. Despite the unusual levels of architectural and sequence similarities, parallels between ROL-3, SEV and ROS functions remained to be established experimentally. Vertebrate homologues of drosophila genes can have conceptually similar functions when compared to their invertebrate counterparts. To help understand the function of ROS in vertebrate species, several groups have conducted detailed ROS expression pattern studies. 4. c-Ros expression in development and adults: Avian and rodent species Expression of ROS was examined in detail in chicken, mouse and rat tissues at various stages of development through adulthood. Northern blot analysis, RNAse protection assays, and in situ hybridization demonstrate that c-Ros is expressed in a spatial, temporal and cell-type specific manner. Expression of c-Ros is found in kidneys,

small intestines, lungs, heart, and male reproductive organs and is highly restricted to epithelial cells. 4.1. c-Ros expression in the kidney and intestinal tract Chicken, mouse and rat kidneys contain moderate levels of c-Ros mRNA in embryos. Levels of c-Ros expression rise during the transition period from development to early postnatal and remain high (chicken and rat) or tapered down (mouse) to low levels in adult kidneys [61–63]. In situ RNA hybridization of chicken kidney at various stages of development demonstrated c-Ros expression to be initially localized to the tips of the collecting ducts [61]. As development progresses, c-Ros expression is detected in the entire epithelial layer of the collecting duct system and progresses to involve the epithelial cells of the larger tubules and even the proximal part of the ureters [61]. This is in contrast to in situ hybridization data obtained from mice where in early embryos, c-Ros expression was confined to the early ureteric bud epithelium as it proliferates and connects with the developing metanephrogenic mesenchyme. As the kidney develops, high levels of c-Ros message are found predominantly at the terminal ends of the budding ureteric epithelium, suggesting a role for ROS in reciprocal epithelial-mesenchymal interactions [41,62,63]. Reciprocal epithelialmesenchymal induction interaction describes the well-established process in kidney development where mesenchymal cells cause the ureters to branch and its epithelial cells to differentiate. At the same time, the differentiating epithelial cells induce the differentiation of mesenchymal cells into epithelial cells of the renal tubules and glomeruli [64]. The expression pattern of c-Ros in the developing kidney coincides with differentiation events driven by epithelialmesenchymal interactions that are essential to major morphological and differentiation events that occur during kidney development. In addition, it has been observed that down regulation of c-Ros expression in an embryonic kidney culture system disrupted the morphology of ureteric bud branches and interfered with development of tubular and glomerular elements [41,65]. These observations lead the authors to postulate that ROS functions in mesenchymal-to-epithelial transition (MET) during kidney development. RNAse protection assay of c-Ros expression in intestinal tissue from mice shows that expression of c-Ros is restricted to epithelial cells and corresponds appropriately to the process of induction induced by epithelial-mesenchymal interaction in the developing intestine. Expression of c-Ros is first detected during embryogenesis and significant levels are exhibited during the neonatal period with relative resolution by postnatal week three [63]. This pattern of cRos expression correlates with the gross differentiation of the epithelium into the various brush border cells. In situ hybridization data show that expression of c-Ros is first detected in epithelial cells of the villi at the stage in gut development when the stratified epithelium begins the process of terminal differentiation into columnar epithelium [62]. 4.2. c-Ros expression in the lung In the lungs, all three species studied reveal different kinetics of c-Ros expression. In mice, the levels of c-Ros are relatively low in embryogenesis, rise slightly after birth then decline and remain low into adulthood [62]. In rats, levels of c-Ros mRNA in the lung are detected during embryogenesis, decrease postnatally and remain constant in the adult [33]. In chickens, levels were shown to increase after birth and remain high in adult tissues [61]. Discrepancies in the pattern of c-Ros expression in the lung between the two rodent species are most likely due to the differences in levels of sensitivity in detection modalities (RNAse protection assays versus northern blot analysis) inherent to the methods utilized. Nevertheless, c-Ros is expressed in adult lungs albeit at relatively low levels.

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In addition, characterization of the transcripts found in rat lungs revealed multiple messages. The predominant message codes for a protein analogous to previously elucidated and characterized c-Ros mRNAs. Interestingly, one lung c-Ros transcript clone contained a 171 base pair insertion just downstream of the transmembrane domain, which is likely to be the result of an alternative splicing event. Significantly, this insertion sequence contains a stop codon, which if translated, would result in a receptor membrane protein without the cytoplasmic tyrosine kinase portion [33]. Further elucidation of this observation is needed especially since this may represent a potential artifact that arose during the cDNA library construction. Confirmation of a ROS splice variant, which creates a membrane-bound receptor uncoupled to kinase activity, would represent an additional mode of functioning for this still elusive receptor. 4.3. c-Ros expression in reproductive tissues In mice, testicular expression of c-Ros is detected only in adults and the transcript has been shown to be approximately 4.5 kb [62,63]. Of note, the length of the testicular transcript is not sufficient to correspond with the full ROS protein and its length differs from the transcripts found in other tissue (8.3 kb). In situ hybridization of adult testes showed c-Ros expression to be present only in mature stages of germ cell development, namely spermatids and spermatozoa [63]. This suggests that the expression of c-Ros may correspond with the onset of male sexual maturity. There are numerous examples of truncated mRNA transcripts in adult testes. The purpose of testicular mRNA variants remains to be established but it has been suggested that alternate transcripts may result in changes in localization, domain composition, or creation of a functionally different protein compared to its somatic cell counterpart [66]. However, the nature of this shortened c-Ros transcript and evidence of ROS protein expression of any form in spermatozoa remain to be determined. In situ hybridization data show c-Ros mRNA present in the caput segment of the epididymis in adult mice. c-Ros expression is restricted to epithelial cells of the epididymis and is initially detected at the onset of regionalization of the caput epididymis [67]. During regionalization, the caput epithelium undergoes terminal differentiation into the various types of epithelial cells necessary for function of the mature epididymis. The importance of ROS in epididymal

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development and function is reflected in the phenotype of ROS-null mice (see section 5) where ROS deficient male mice are unable to reproduce due to defective sperm function as a result of improper spermatocyte capacitation, a process dependant on a fully functional epididymal epithelia [67]. 5. c-ROS expression in humans There has been very limited data reported on c-ROS expression in humans. The finding of c-Ros expression in the proximal murine epididymis along with subsequent studies showing c-Ros knockout mice to be healthy but infertile has inspired investigations into the expression of c-ROS in human epididymis. The rationale for these experiments is the concept of developing a male contraceptive based on inhibition of ROS function. Expression of c-ROS in the human epididymis as analyzed by RT-PCR and in situ hybridization demonstrated that ROS is present throughout the human epididymis and expression levels varied among epididymal segments and is absent from the proximal caput [68]. This is in stark contrast to what is observed in mice where cRos expression is restricted exclusively to the caput epididymis. In order to consider ROS inhibitors as contraceptive agents, evidence demonstrating that inhibition of ROS function (through genetic or chemical methods) in adult animals (i.e. after the differentiation program has taken place) results in diminished sperm capacitation is an absolute necessity. Interestingly, immunohistochemical staining indicates that ROS is localized to the cytoplasm of basal cells and in the supranuclear cytoplasmic compartment of principal cells within the epididymal corpus [68]. This is a surprising observation given that full-length ROS protein is a membrane-bound receptor. To gain insight into c-ROS expression in human, we have performed a northern blot analysis on RNA isolated from various adult human organs and determined that c-ROS expression is detectable mostly in the lungs (Fig. 2A). Size variants are also detected in RNA isolated from placenta and skeletal muscle tissues. Although very informative vis-à-vis transcript sizes, northern blots notoriously suffers from low sensitivity of detection for low abundant messages. This drawback is overcome with the advent of microarray gene expression studies where the expression patterns of any given gene is readily ascertained from various sources. This very informative, quantitative type of in silico analysis complement expression

Fig. 2. Expression of c-ROS mRNA in adult H. sapiens tissues. (A) Northern blot analysis of adult human tissues using a radiolabeled cDNA probe representing c-ROS full-length cDNA. Different length messages are observed in placenta, lung and skeletal muscle. (B) Relative expression levels of c-ROS gene extracted from gene expression profiling of mRNA isolated from multiple adult human tissues [69]. The dot represent the average of multiple mRNA samples for a given tissue and the vertical bars indicate the highs and lows values for each group.

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information obtained from northern blot analyses. We mined human expression array databases for c-ROS expression and consistently found that the highest levels of c-ROS expression in adults is in lung tissues (Fig. 2B) [69]. The development of high affinity antibodies capable of detecting ROS protein in immunohistochemistry-based analyses of human tissues would greatly facilitate the elucidation of ROS function in these tissues. Overall, expression of c-ROS is found to be both temporally and spatially regulated. Its patterned localization of expression during embryogenesis and in various adult organs suggests that ROS may play a role in the mature function of these organ systems beyond the developmental role of epithelial-mesenchymal interactions. Parallels in ROS function between the drosophila photoreceptor R7 cell differentiation and the various tissues where c-ROS is expressed at time of terminal differentiation strongly suggest that ROS initiates signaling events that are key component of a differentiation program of epithelial tissues. 6. c-ROS Expression in disease

carcinomas and adenocarcinomas showed a three-fold increase in cROS expression compared to normal lung tissue. c-ROS expression was elevated in both early and late stage lung tumors suggesting that ROS is important for lung tumor development and/or maintenance rather than progression [75]. Elevated c-Ros expression is also observed in non-small cell lung cancer (NSCLC) tumors in an oncogenic Kras initiated mouse model of lung cancer [76,77]. This suggests that in these tumors, ROS may work in unison with activated K-RAS to establish signaling pathways with transforming activities. Increasing evidence shows that deregulated expression of c-ROS may be important to the pathogenesis of human lung cancer. Several microarray analyses of tumor specimens revealed significantly elevated c-ROS expression levels in 20 to 30% of patients with NSCLC [78–80]. Using hierarchical clustering, one study demonstrated that elevated c-ROS expression was part of a molecular signature of lung adenocarcinoma subclasses, which is associated with well-differentiated tumors with a more favorable outcome [78]. 6.3. Breast, stomach, liver, kidney and colon cancers

6.1. Cancers of the central nervous system Early studies on the expression of human c-ROS in cancers pointed to events leading to the discovery of aberrant expression in tumors of the CNS, specifically gliomas (Table 1). A survey of 45 human cell lines from various normal and cancer tissues by RNase protection assays, revealed c-ROS transcript expression in 56% of the glioblastoma cell lines analyzed ranging from 10 to 60 transcripts per cell [70]. In these experiments, c-ROS expression was not detected in normal brain tissue or in cell lines from 16 other types of cancer. Southern blot analysis showed that the c-ROS gene is present in normal copy number making gene amplification an unlikely event to account for the observed aberrant expression. This suggests that ectopic expression of c-ROS, without gene amplification, may contribute to tumorigenesis. Two independent analyses of surgical specimens for c-ROS expression by RNase protection and cDNA hybridization techniques have yielded similar results. High levels of c-ROS expression in 33% and 40% of glioblastoma surgical tumors has been reported [71,72]. The lack of c-ROS detection in lower grade astrocytomas suggests that expression of c-ROS may be an event important for tumor progression [71]. However, there has been no definitive study performed demonstrating a prognostic value to c-ROS expression in gliomas. In other studies, high levels of c-ROS transcripts were detected in meningioma tumor specimens [73,74], indicating that ectopic c-ROS expression in different cell types can give rise to histopathologically distinct tumors. Combined, these studies clearly demonstrate that c-ROS is aberrantly expressed in 33% to 56% of glioblastoma tumors and up to 55% of meningeal tumors. These observations most certainly warrant further investigation into the potential role(s) that ROS play in gliomagenesis. 6.2. Lung cancer Aberrant expression of c-ROS was also detected in cancerous lung tissue in mice. Microarray analysis of carcinogen-induced murine lung

Table 1 c-ROS expression in cancers of the CNS Tissue

Expression (%)

References

GBM-cell lines GBM-tumors GBM-tumors Astrocytoma-tumors (WHO grade II) Anaplastic astrocytoma tumors (WHO grade III) Meningeal tumors

56% (5/9) 33% (2/6) 40% (4/10) 0% (0/5) 0% (0/4) 55% (16/29) 21% (4/19)

[70] [71] [72] [71] [71] [73] [74]

In a gene expression profile survey of 20 fibroadenomas of the breast, a common benign tumor, c-ROS was found overexpressed at levels more than two-fold higher than normal tissues [81]. Fibroadenoma is the most common types of benign breast tumor in young women and patients with such tumors have a two-fold increase in the relative risk to develop malignant breast cancer. It is thought to arise from a disrupted developmental program. It is conceivable that overexpression of c-ROS in these tumors may be attributable to a role in tumorigenesis as a function of improper developmental processes. This research is certainly worth pursuing especially in the context of ROS function in the various differentiation programs mentioned above. It has recently been shown in a carcinogen-induced rat stomach cancer model that c-Ros expression, along with a handful of other genes, is induced upon carcinogen treatment and this expression persists long after chemical exposure [82]. This led the authors to suggest that such persistent changes, of which ROS figures prominently, are drivers of tumorigenesis. Similarly, Yovchev et al., recently discovered that de novo c-Ros expression is associated with induction of hepatic progenitor cells activation in a rat model of liver injury and that c-Ros expression is high in a rat hepatoma cell line [83,84]. These observations further the need to address the function of ROS signaling in the context of cell differentiation and transformation. More recently, a global sequencing survey of all tyrosine kinases in 254 established cell lines uncovered three new ROS mutations in two colon adenocarcinoma and one kidney carcinoma cell lines [85]. Interestingly, the two colon carcinoma mutations in c-ROS result in exon skipping which lead the authors to suggest that translation of these cROS species are prematurely terminated and are predicted to give rise to secreted (non-membrane bound) form of the receptor. This raises an interesting concept worth pursuing especially with regards to ligand identification. Unfortunately, the authors did not address the causative nature of these observations. Given the fact that both cell lines are known to contain chromosomal rearrangements at 6q22 (http://www. ncbi.nlm.nih.gov) where c-ROS is located [86-88] (http://www. ensembl.org/Homo_sapiens/contigview?l=6:117716223-117853711), it is quite possible that these observed ROS abberations are the results of translocations and/or deletions which begs the question, what is the status of the carboxyl terminal kinase domain of ROS in these cell lines? Collectively, these observations highlight the importance of elucidating ROS function in cancer genesis and maintenance especially in the context of co-expression with other activated oncogenes. The use of animal models, which offer genetically tractable platforms, will certainly advance our understanding of ROS function in tumor initiation and progression. It is interesting to draw parallels with the

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drosophila SEV role in terminal differentiation during development and what is observed in human lung adenocarcinomas. At which point and under what circumstances, if any, does c-ROS expression trigger differentiation cues during the transformation process? These questions need to be addressed experimentally and the results will certainly further our understanding of cancer especially in the context of developing therapeutically active ROS-specific kinase inhibitors. 6.4. Heart disease Recent studies have suggested a link between specific c-ROS alleles and heart disease. A gene-centric association study of more than 11,000 single nucleotide polymorphisms (SNPs) spanning approximately 7,000 genes uncovered a c-ROS allelic variant that had statistically significant association with myocardial infarctions (MI) [89]. These results were directly confirmed in a prospective population based study where an increased risk for MI was associated with the same variant c-ROS genotype identified by Schiffman et al. [90]. Interestingly, a recent retrospective study [91] failed to validate these studies. This seeming discrepancy emphasizes the need for further investigations to verify the applicability of c-ROS polymorphisms to cardiovascular medicine. A separate study showed that the same cROS SNP is significantly associated with the incidence of restenosis after coronary stenting [92]. More recently, a similar association study has linked a c-ROS polymorphism to increased blood pressure and a factor in the development of hypertension in Japanese individuals [93]. Assuming that this SNP is functionally associated to the c-ROS gene rather than other distant genes, the suggestion that ROS may play a role in cardiac function is further solidified by the nature of the c-ROS polymorphism studied. This SNP is a coding polymorphism changing amino acid residue 2213 of ROS from asparagine to aspartic

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acid. This residue is located immediately next to the kinase domain of the receptor, which allows one to speculate that these two allelic variants of ROS may be endowed with different intrinsic kinase activities. However, the location and levels of c-ROS expression in cardiac tissues still remain to be determined and should be the focus of intense research. Once these basics are established, experiments aimed at obtaining mechanistic insights into the manner in which these alleles affect ROS function may lead to a better understanding of the role of ROS in heart disease. 7. Oncogenic ROS fusion kinases Cancer is an opportunistic disease. Throughout its progression, individual tumor cells select for genetic events, which gives them net growth and survival advantages. Chromosomal rearrangements in the form of translocations, deletions and amplifications are very commonly observed in tumors. This is because, all too often, the result of these events is the creation of gene fusion with potent oncogenic activities. Over 35 distinct members from ten RTK families have been observed in such chromosomal rearrangements in various kinds of malignancies (see Fig. 3). For the most part, these genomic events lead to the juxtaposition of novel sequences to those coding for the kinase domain of a given RTK. Production of fusion kinases in this manner result in chimeric kinases that are ligand independent and constitutively activated counterparts to their ligand-regulated full-length wild-type receptors. With the exception of ROS fusion kinases (see below), all known fusion partners contain protein dimerization domains, which force the fusion kinase monomers into homodimers leading to a constitutively activated kinase state. In addition, these fusion kinases are cytosolic, which preclude their entry in the endocytic pathway and therefore, their lysosomal targeting and

Fig. 3. Receptor tyrosine fusion kinases in cancer. Schematic representation of the ten RTKs known to be involved in chromosomal events (translocations and deletions), which create fusion chimeric oncogenic kinases in mammalian cancers. The 5′ fusion partner genes for each receptor are indicated in red.

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degradation thus escaping their normal mechanisms of downregulation. Creation of these fusion kinases leads to unchecked signaling events and cellular transformation ensues. 7.1. V-ROS The oncogenic DNA of the avian sarcoma virus UR2 is a rearranged product of the chicken c-Ros gene. Comparison of the UR2 v-ros and Gallus gallus c-Ros genes revealed structural differences in p68v-ros that are important mediators of oncogenic transformation (Fig. 4): (i) v-ros is joined in frame to the gag sequence of UR2 seven amino acids upstream of the TM domain of c-Ros, (ii) there is a three amino acid insertion within the TM domain of v-ros and (iii) c-Ros sequences coding for the 3′ carboxyl terminal tail are truncated in v-ros. Structure activity relationship experiments demonstrated that the TM domain of p68v-ros is functional and that targeting of p68v-ros to the plasma membrane is necessary for the transformation of CEFs by UR2 [23,94]. Moreover, it was shown that the p19gag sequence does not mediate oligomerization of the p68v-ros protein at the plasma membrane, leading the authors to suggest that membrane targeting rather than homodimerization was sufficient for transformation [22,95]. Further work provided insight into the significance of membrane targeting for cellular transformation. First, the additional three amino acid residues within the TM domain of p68v-ros proved to be necessary for the transformation activity of v-ros [96] and second, chimeric EGFR-ROS receptors differing only by their TM domains demonstrated that recycling modalities, signaling pathways activation and ensuing cell transformation activities were significantly dictated by sequences within the TM domain [97]. Finally, swapping of the 3′ carboxyl terminal tail sequence of v-ROS to that of c-ROS, which essentially add an autophosphorylation site, does not modify the

transforming potential of p68v-ros [98]. A great deal of information pertaining to ROS-mediated mechanisms of cellular transformation transpired from the seminal biochemical work on UR2′s p68v-ros. This information paved the way to a better understanding of ROS signaling networks induced during transformation. 7.2. FIG-ROS Our search for fusion kinases in primary brain cancers led us to characterize an aberrant c-ROS transcript in glioblastoma cell lines [30]. We have shown that this transcript is the result of a small intrachromosomal deletion that fuses a then uncharacterized gene that we termed FIG (Fused in Glioblastoma) to c-ROS sequence (Fig. 4) [29]. FIG is a membrane-bound protein that localizes to the Golgi apparatus [29]. The FIG-ROS transcript, which is encoded by seven FIG exons and nine ROS exons, also localizes to the Golgi apparatus. We investigated the biological activities of the FIG-ROS fusion protein and found that FIG-ROS is a potent oncogene with a unique mechanism of tyrosine kinase activation. We demonstrated that it is this localization to endomembranes that is responsible for the oncogenic potential of FIGROS [28]. Finally, expression of FIG-ROS in adult CNS readily induces glioblastoma formation in vivo in a genetically engineered mouse model of brain cancer [31]. 7.3. SLC34A2-ROS and CD74-ROS A recent large-scale survey of tyrosine kinase activity in lung cancer uncovered two novel ROS fusion proteins associated with NSCLC [32]. Analogous to p68v-ros in UR2 ASV and FIG-ROS in glioblastoma cells, ROS is activated by genetic rearrangements that result in fusion proteins between the membrane-associated protein

Fig. 4. ROS fusion kinases. Schematic representation of the genomic loci of all known ROS fusion events and their corresponding resulting protein fusion products (except for mcf3). Notice that all rearrangements occurred in the same genomic vicinity in the ROS gene. ROS's transmembrane (TM) domain is encoded by exons 35 and 36. With the exception of FIGROS, all ROS fusion kinases are predicted to be plasma membrane bound. The SLC34A2-ROS fusion gives rise to two transcripts through alternative splicing events. The predicted SLC34A2-ROS and CD74-ROS fusion proteins are drawn as bi-membrane spanning receptors, a topology not yet confirmed experimentally.

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solute carrier SLC34A2 and the type II transmembrane protein CD74 to ROS extracellular juxtamembrane domain (Fig. 4). SLC34A2 is a member of the sodium phosphate cotransporter solute carrier group of membrane transport proteins (for a review see [99]). Members of the SLC34 family are topologically arranged as eight TM domain receptors with intracellular NH2- and COOH-termini [100]. SLC34A2 is expressed in the small intestine, lung, testis, liver and secreting mammary gland where it plays a role in the homeostasis of inorganic phosphate [101–103]. In the lung, SLC34A2 is localized at the apical pole of alveolar type II epithelial cells [104]. In HCC78 cells, a translocation between chromosomes 4p15 and 6q22 causes an in frame fusion of exon four of SLC34A2 to exons 33 and 35 of ROS, the latter being the result of an alternative splicing event skipping exons 33 and 34 (Fig. 4). The SLC34A2-ROS protein has been shown to localize to membrane fractions and display a constitutive kinase activity. siRNA-mediated downregulation of SLC34A2-ROS-induced apoptosis in transformed cells demonstrating that ROS signaling is essential for survival of these NSCLC cells [32]. CD74 is an integral membrane protein shown to function as the receptor for the macrophage migration inhibitory factor (MIF) [105]. CD74 is also known as an MHC class II-associated invariant chain that plays a critical role during peptide presentation to CD4-positive lymphocytes [106]. CD74 is ubiquitously expressed and there seems to be increased levels of CD74 at the surface of multiple malignant cells [107–114]. A t(5;6)(q32;q22) translocation in a NSCLC tumor from a patient gives rise to an in frame fusion of exon six of CD74 to exon 35 of ROS (Fig. 4). It is of interest to decipher the topology of both SLC34A2-ROS and CD74-ROS given the presence of two TM domains in each fusion kinase. Regardless of the final topology, the kinase domain of ROS is predicted to remain intracellular (Fig. 4). Detailed biochemical experimentations are necessary and worth pursuing in order to determine the membrane insertion topology for these ROS fusion proteins. Interestingly, unlike most other fusion RTKs, all fusion ROS kinases discovered so far are membrane bound. Also, these genomic rearrangements occur near ROS exon 35, which codes for the TM domain (Fig. 4). This observation perhaps underlines the presence of a hotspot for genomic rearrangements within the ROS locus. Thus far, all four chimeric ROS proteins identified have fusion partners with the capacity for membrane localization. Many RTKs activated by genomic rearrangements have several different fusion partners. Therefore, it is not surprising to observe multiple ROS fusion events and it is conceivable that additional oncogenic ROS fusion kinases exist in other human cancers. It is becoming increasingly evident that activation of ROS kinase by genetic rearrangement is an important event in human cancers. It will be interesting to compare the signaling effector proteins that are activated by all three mammalian ROS rearrangement products in NSCLC and glioblastoma. It remains to be seen whether additional and/or different signaling events specifically emanate from these fusion kinases when compared to full-length ligand-regulated ROS. If so, determining these differences in pathway utilization would have profound value in terms of cancer therapeutics development. 8. Epithelial differentiation; insights into the phenotype of ROS-null mice To gain a better understanding of ROS function, mice with a deletion of the c-Ros gene were generated and characterized [67]. The mutation resulted in a gender and tissue-specific defect. Female homozygous ROS-null mice do not display any detectable abnormalities nor do heterozygous mice of either sex. Homozygous ROS-null male mice, however, are infertile but otherwise healthy. Further studies demonstrated that ROS is not needed for sperm production or sperm function as evidenced by investigations using chimeric mice consisting of wild type and homozygous mutant ES cells. In these

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chimeric mice, precursor germ cells are able to produce functional sperm when given a wild-type environment. Moreover, spermatocytes isolated from homozygous males are able to fertilize in vitro demonstrating that the infertility present in nullizygous c-Ros males is not spermatocyte cell autonomous. Further studies demonstrated that the source of infertility lies in the improper maturation of the spermatocytes as they migrate through the convoluted network of tubules of the epididymis, a process known as capacitation. Capacitation of immature to mature spermatozoa requires a well-balanced osmolarity and composition of the luminal epididymal fluid, which in turn is regulated by the highly secretory epithelial layer of the epididymis (for a review see [115]). At puberty, answering to the emergence of androgen production, the cuboidal epithelia of the proximal region or caput of the epididymis differentiates into tall columnar epithelia and gains enhanced secretory features. This process coincides with the expression of c-Ros. Not surprisingly, it has been shown that c-Ros expression is responsive to androgens [116]. In ROS-null homozygous male mice, this differentiation process is absent which leads to a failure of the proximal epithelia to function normally and result in an imbalance in the osmolarity and composition of the luminal fluid with consequences of improper spermatocyte maturation [117–121]. This suggests that at the onset of puberty, androgens trigger a gene expression profile of which c-Ros is part and whose signaling plays a key role in the program of epididymal terminal differentiation. As additional research into this area continues, it will be interesting to compare the signaling components of ROS that are involved in the epididymal differentiation program to those observed in the various cancers where ROS is thought to play a role. Using this tractable system to establish valuable parallels between these systems will tremendously advance our knowledge of ROS function. Insights into ROS signaling pathways involved in terminal differentiation of the caput epididymis came from the observation that, analogous to ROS-null animals, male mice carrying the “viable motheaten” (mev) mutation are also sterile due to a similar defect in differentiation of the epididymis which result in impaired sperm maturation [122]. This observation suggested that ROS and SHP-1 function in signaling pathways responsible for terminal differentiation of the caput epithelia. mev is a naturally occurring splice site mutation in the gene coding for the SH2 domain protein tyrosine phosphatase SHP-1. The mev allele is hypomorphic and codes for two SHP-1 protein variants both with aberrant phosphatase domains, one with a small deletion and the other with a small insertion [123]. Together, these mutant proteins have been shown to retain approximately 20% of wild-type SHP-1 phosphatase activity [124]. SHP-1 activity was confirmed to be coupled to ROS signaling when it was demonstrated that SHP-1 (Ptpn6) is co expressed with c-Ros in the caput epididymis of adult mice and elevated phosphorylation of ROS was observed in the epididymis of mev mice. Furthermore, in vitro biochemical experiments demonstrated that SHP-1 directly binds autophosphorylated ROS via an interaction between the aminoterminal SH2 domain of SHP-1 and a autophosphorylated phosphotyrosine residue in the carboxyl terminal end of ROS [122]. It is known that engagement of the SH2 domain of SHP-1 activates its catalytic activity and thus, physical interaction between ROS and SHP-1 is thought to activate the phosphatase activity of SHP-1. These studies suggest that SHP-1 activation is under the control of ROS signaling in vivo and that activated SHP-1 triggers or leads to a differentiation program. Parallel observations from the mev mice hematopoietic system has led to the dogma that SHP-1 acts as a negative regulator of RTKs, cytokine and chemokine receptors and integrin signaling [125]. Given the information at hand, it appears that in epididymal tissues, SHP-1 acts as a positive effector of the RTK ROS since inactivation of either ROS or SHP-1 independently result in the same phenotype, that is, failure of the epididymal epithelia to differentiate. It has recently been shown in epithelial cells that SHP-1 can act as a positive

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regulator of RTK signaling [126]. It seems that the results of SHP-1 engagement upon growth factor receptor activation is context dependent. During development of the epididymis, the evidences suggest that SHP-1 activation is a pro-differentiation event. Perhaps, in different context, engaging SHP-1 may lead to an alternative outcome. Regardless, the studies conducted in both c-Ros knockout mice and viable motheaten mice have demonstrated the importance of regulating ROS signaling for the proper differentiation and development of the caput epididymis.

receptor [97] or NGF receptor [40,128] are fused to the intracellular kinase domain of ROS thus rendering ROS activity ligand-regulated. These ligand-regulated ROS chimeric species were shown to activate several signaling pathways. It has been demonstrated that ROS activity can lead to activation of the SH2 domain tyrosine phosphatases SHP-1 and SHP-2, the mitogen-activated protein kinase ERK1/2, insuline receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), STAT3 and VAV3 signaling pathways (Fig. 6) [31,97,129,130].

9. ROS elicits signal transduction programs of proliferative growth and cell survival

9.1. SH2 Domain containing tyrosine phosphatases SHP-1 and SHP-2

ROS activates several signaling pathways that are known to be important for cell growth and survival. Activation of these pathways is mediated by the kinase activity of ROS on specific substrates and by the formation of phosphotyrosine recruitment sites within ROS's Cterminal tail. Fig. 5 schematically depicts the tyrosine residues of the juxtamembrane domain and the C-terminal tail of V-ROS and C-ROS. Interspecies comparisons reveal the conservation of tyrosine residues during evolution. In mammals, tyrosine residues 2274 and 2334 have been shown to be phosphorylated sites and substitution of both residues to phenylalanine abrogate the transforming activity of oncogenic ROS [28]. The study of ROS-induced signaling pathways and transformation continues to be hampered by the absence of a known ligand for c-ROS and the inability to overexpress full-length wild-type c-ROS in different types of mammalian cells. A simple strategy employed over the years to overcome these obstacles has been the development and utilization of ROS chimeric receptors where the extracellular ectodomain of ligand-activated RTKs such as the Insulin receptor [127], EGF

In addition to the aforementioned activation of SHP-1 in the differentiation program of the epididymal epithelia, ROS has been shown to interact with, phosphorylate and activate the SH2 domain containing tyrosine phosphatase SHP-2 [31]. Germ line missense mutations of PTPN11, the gene coding for SHP-2, are responsible for the majority of Noonan syndrome cases, an autosomal dominant disorder characterized by a short stature, typical facial dysmorphology, congenital heart defects and associated with an increased incidence of leukemia [125,131,132]. In addition, several somatic Ptpn11 mutations have been reported in a high percentage of hematological malignancies [133–136]. Somatic mutations in Ptpn11 have also been found in solid tumors of the colon, lungs, skin and peripheral nervous system [137]. All of these PtpN11 mutations predict an activated form of SHP-2, which strongly suggest that constitutive activation of SHP-2 is an oncogenic event. Consistent with this concept, transduction of mouse bone marrow cells with a constitutively activated SHP-2 protein cause a fatal JMML-like disorder in vivo [138]. Constitutively active SHP-2 is considered an oncogene since it is known that SHP-2 positively regulates the ability of several

Fig. 5. Comparison of ROS intracellular tyrosine residues in different species. Schematic representation of the relative positions of intracellular tyrosine residues of V-ROS, G. gallus and H. sapiens C-ROS. Excluded from the schematics are those tyrosine residues within the kinase domain. Notice the sequence conservation of the juxtamembrane (Y1923) and Cterminal (Y2274) of human ROS when compared to those of chicken ROS. Notice also the truncation of the C-terminal tail, which includes Y2289, in V-ROS. This suggests that phosphorylation of this site, within the context of V-ROS, is not a requirement for UR2-mediated cell transformation. However, this site, along with what appears as an evolutionary duplicated site Y2334, are necessary for transformation in mammals. Figure not drawn to scale.

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Fig. 6. Schematic representation of signaling pathways activated by ROS. The map is an amalgamation of observations obtained from chimeric EGFR-ROS, TRK-ROS membrane-bound receptors expressed in fibroblasts as well as V-ROS and FIG-ROS derived signaling events. Putative ligand(s) are depicted as soluble molecules and hypothetical activation of ROS through contact with ECM proteins is also indicated. Not represented is the potential G-protein coupled receptor GPRC5B on adjacent cells. ROS autophosphorylation sites are docking sites for the tyrosine phosphatases SHP-1 and SHP-2. ROS activates PI3K through the scaffolding proteins IRS-1 and/or GAB1. Activated PI3K in turn activates an AKT/mTOR signaling axis. STAT3 and VAV3 are phosphorylated directly by ROS kinase activity.

RTKs to activate the Ras/Raf/MAPK signaling cascade [139–144]. In most cases, the catalytic activity of SHP-2 is required to propagate receptor-mediated signaling to MAPK [145,146]. Further biochemical and genetic evidences of SHP-2 acting as a positive regulator of growth factor receptors come from studies on D. melanogaster SHP-2 homologue corkscrew (csw). The oncogenic activity of FIG-ROS in fibroblasts is mediated by an interaction with and the phosphorylation of SHP-2, suggesting that activation of SHP-2 is a crucial event in FIG-ROS transformation [31]. Interestingly, phosphopeptides corresponding to the tyrosyl phosphorylation sites of SHP-2 were recovered in tumor samples and cell lines that contained the rearranged SLC34A2-ROS and CD74-ROS products [32] suggesting that constitutively active ROS may preferentially signal through the SHP-2 tyrosine phosphatase. The remaining question is, what are then the signaling events downstream of SHP-2 activation? Perhaps the recently identified link between SPROUTY proteins and SHP-2 activity will shed light on SHP-2 signaling pathways [147]. 9.2. MAP kinase Furthering the homology between SEV and ROS, analysis of ligand stimulated EGFR-ROS and TRKA-ROS chimeric receptors demonstrated a temporal activation of ERK1/2 in NIH-3T3 cells [97,122]. Similarly, oncogenic V-ROS also activates ERK1/2 in chicken embryonic fibroblasts (CEFs) [130]. Surprisingly, chemical inhibition of the MAPK pathway by the MEK inhibitor PD98059 had no effect on the transforming potency of chimeric EGFR-ROS on CEFs or NIH-3T3 cells [148]. In addition, in FIG-ROS-induced transformed fibroblasts, elevated levels of phosphorylated ERK1/2 could not be detected (Charest, A. unpublished data). Taken together, these observations suggest that, although activated under certain circumstances, the MAPK pathway may play little, if any, role in the transformation of these cell types by activated ROS. Fibroblasts expressing either V-ROS or FIG-ROS are capable of growing in an anchorage-independent fashion, a feature intrinsic to many cancer cells. It is known that powerful reorganization of

cytoskeletal structures is necessary for this kind of morphological transformation. Not surprisingly, V-ROS expressing cells demonstrated phosphorylated versions of a series of cytoskeletal proteins involved in the formation of focal adhesions and cell-cell interactions [130] suggesting that constitutively activated ROS can signal to elicit structural rearrangements of the cytoskeleton as well as modulate biochemical events necessary for cell-cell interactions. It remains to be determined if wild-type ROS signaling is also capable to modulate cellECM and cell-cell interactions especially in the context of epithelial differentiation as seen in the epididymis, small intestine and ureteric buds. 9.3. The PI3K and Akt pathway The PI3K signaling pathway promotes growth factor-mediated cell survival in many cell types. PI3K also has key regulatory functions in cellular proliferation, differentiation, apoptosis, glucose metabolism and vesicle trafficking. PI3K, a lipid kinase, is activated by RTKs through recruitment at the plasma membrane, a process fulfilled by the engagement of the SH2 domain of its p85α regulatory subunit to phosphorylated tyrosine residues on the receptors themselves or on scaffold proteins such as IRS-1 or GAB-1 [149]. This binding then elicits an activation of PI3K catalytic subunit, which, in turn phosphorylates inositol lipids to produce various phosphatidylinositol phosphates (reviewed in [150]). The latter serve as second messengers to recruit and activate a multitude of proteins that contain pleckstrin homology domains or FYVE fingers. Among these, several kinases (PDK1, PDK2 and Akt/PKB), nucleotide exchange factors (TIAM1, VAV and SOS), GTPase-activating proteins and phospholipases have been shown to respond to PI3K activity. The AKT kinase is a major downstream target of PI3K activation and has functions both in cell growth and survival (for reviews see [151,152]). The list of AKT substrates is ever growing and comprises substrates that have been shown to be involved in cellular growth, survival and metabolism (for reviews see [153–156]). Activation of the PI3K/AKT pathway has been demonstrated to be an important event for ROS-induced transformation. Chemical and genetic inhibition of the PI3K pathway significantly attenuated the

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ability of activated ROS to induce colony formation and anchorageindependent growth of fibroblasts [148,157]. In these cells, activation of PI3K by ROS is thought to be mediated by the scaffold protein IRS-1 since an elevated PI3K activity is immunoprecipitated using anti IRS-1 antibodies in V-ROS expressing CEFs [130,157]. Activation of AKT is frequently associated with malignant astrocytomas in humans and hyperactivation of its downstream effector, mTOR, is found human and mouse brain cancer [158–161]. We have shown that the oncogenic fusion protein FIG-ROS can activate a PI3K/AKT/mTOR signaling axis in FIG-ROS-induced brain tumors [31]. In addition, the mTOR inhibitor, rapamycin, induced dose dependent growth inhibition in FIG-ROS brain tumor cell cultures [31], which suggest that mTOR inhibitors may be effective in treating malignancies harboring, activated ROS. Although detailed mechanistic features of ROS-mediated PI3K activation remain to be established in different cell types, the data reported in the literature strongly support the view that ROS signals through a PI3K/AKT axis. 9.4. STAT3 Activation Signal transducers and activators of transcription (STATs) are transcription factors involved in cytokine and growth factor receptor induced gene expression. STATs are activated by tyrosine phosphorylation, which result in their dimerization and translocation to the nucleus where they regulate the transcription of target genes by binding to specific DNA-response elements. STATs have been found to be activated in a wide variety of tumors and in oncogene-transformed cell lines (reviewed in [162,163]). It is becoming increasingly clear that STAT3 in particular, is constitutively activated in oncogenic tyrosine kinase transformed cells and in certain instances shown to be essential for their transformation activity [164,165]. Interestingly, constitutive activation of STAT3 has been reported in malignant brain cancer tumors with overexpression of the EGF receptor [166]. Many STAT3 transcriptional target genes are anti-apoptotic, for example survivin, MCL-1, BCL-x and BCL-2 have been shown to be transcriptionally responsive to STAT3 activation. Not surprisingly, STAT3 activation has been associated with resistance to chemo- and radiation therapy, a salient feature of malignant brain cancer. Stimulation of STAT3 signaling is an additional event for oncogenic transformation by activated ROS in fibroblasts [167]. It has been shown that activation of STAT3 is required for ROS-mediated transformation in vitro. Expression of dominant negative STAT3 partially suppressed ROS-induced anchorage-independent growth of NIH-3T3 cells and was shown to inhibit both initiation and maintenance of the transformed state [148,167]. The transcriptional program initiated by a ROS-STAT3 signal axis in these cells remains to be established and is certainly worth pursuing in other tissues especially in the context of changes in gene expression profiles that are associated with ROS-mediated anchorage-independent growth. 9.5. VAV3 Cytoskeletal changes brought about by V-ROS-mediated transformation were observed very early. UR2 transformed cells present and exaggerated fusiform appearance, which suggested that persistent ROS activity in these cells result in major cytoskeletal effects. VAV3 has been shown to be another mediator of ROS signaling. VAV3 is a member of the VAV family of phosphorylation-dependent guanine exchange nucleotide factors (GEF) for the Rho/Rac family of GTPases. Rho/Rac family GTPases are important regulators of the actin cytoskeleton and are associated with motility and invasion of cancerous cells [168]. The VAVs are therefore connecting stimulated membrane receptors to cytoskeletal reorganization [169]. VAV3 was found to interact with ROS in a yeast two-hybrid screen using the Cterminal end of ROS as a bait [129]. Interestingly, the ROS-VAV3 interaction was shown to be constitutive and independent of ROS

activation, and stimulation of ROS resulted in VAV3 phosphorylation [129]. Nguyen et al. showed that inhibition of RHO family GTPases suppresses ROS-induced colony formation in CEFs and anchorageindependent growth in NIH-3T3 cells [148]. These results are in accordance with the demonstration that RHHO GTPase signaling is necessary for the oncogenicity of oncogenes derived from RTKs such as EGFR, IGFR, MET or RET [170,171]. However, it remains to be determined if VAV3 bridges the RHO family GTPases-induced changes in adhesion upon ROS activation. 10. Ligand(s) The most obstructing element of ROS research has been the inability to identify ROS's natural ligand(s). The high degree of parallelism between ROS and SEV suggest that a mammalian homologue of BOSS would represent a valid candidate. Unlike other receptors whose ligands are conserved throughout evolution, there is a lack of BOSS conservation in more distantly related genomes. This may be the result of an evolutionary replacement of SEV ligands. Sequence alignment of BOSS to mammalian genomes revealed the presence of a single orphan seven transmembrane G protein coupled receptor gene with weak overall homology to BOSS [172]. GPRC5B is related to the metabotropic glutamate receptor family [173,174]. GPRC5B is expressed in the peripheral and central nervous system, in the kidney, pancreas, testis [173-175] and in the placenta and yolk sac during gestation in mice [176]. The expression of this receptor in kidney and testis is interesting in the context of ROS expression and function in these tissues. Experiments, aimed at validating the potential for GPRC5B to activate ROS should be performed. Another source for the search of a putative ligand is the testicular lumicrine space. Given ROS function in the differentiation of the proximal caput epididymis, one can conceive that ligands, produced within the testes, are present within the testicular fluid flowing through the epididymis and referred to as lumicrine factors [115,177]. Interestingly, prevention of testicular exocrine secretion in mice result in regression of the initial segment of the epididymis, similar to the cRos knock out phenotype, suggesting that factors within this fluid act upon ROS as ligands [177]. These observations suggest that the testicular exocrine fluid may harbor molecules capable of activating ROS and thus represent a valid source of material to search for ligand species. Finally, it is quite possible that ROS has more than one ligand. Experiments aimed at elucidating the identity of ROS ligand(s) are deeply needed if we are to fully understand the role and biological significance of ROS activation in normalcy and disease. 11. Conclusion Since its discovery over 25 years ago, research on ROS has been dampened by the lack of a known ligand and the inability to express the full-length wild-type receptor both in vivo and in different cell types in vitro. Nevertheless, a great deal of information gravitates ROS. Genetic screens in invertebrate models led to the deciphering of many signaling components of ROS. Biochemical approaches in mammals furthered our knowledge on how ROS triggers specific pathways, which ultimately leads to cellular transformation and cancer. There are however, many more questions to be answered. What function does ROS play in intestinal and urological tissue development? Is there a role for ROS in cardiac function and if so, what are the biological effect(s) of various c-ROS alleles in this system? Is the aberrant expression of full length, wild-type ROS observed in human cancers a causative or by-stander phenomenon? No doubt the field is at a standpoint, awaiting the identification of a ligand and the development of the necessary tools to address these questions. For example, the creation of a Cre/Lox-based conditional knockout or overexpressing transgenic mouse strains would certainly benefit and undoubtedly advance ROS research. The recent publications on

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