From Mendelian to molecular genetics: the Xiphophorus melanoma model

In human tumor biopsies it is almost impossible to pinpoint the particular molecular abnormalities that determine neoplasia. In animal models where tumorigenesis is initiated by clearly defined genetic events, it is possible to study the genes and their functions that make a normal cell become a fully malignant cancer cell. In the fish Xiphophorus, melanoma can be initiated by simple crossings, and the signaling pathways that govern tumor growth and progression can be delineated. This model offers the prospect of obtaining a complete picture of the molecular changes and regulatory networks underlying tumor formation, which should contribute to a better understanding of some general principles of cancer biology, and identify new targets for melanoma research in particular.     

Interaction of Xiphophorus and murine Fyn with focal adhesion kinase

The Src family kinase/Focal Adhesion Kinase (FAK) complex is a signaling platform playing a crucial role in transformation downstream of oncogenic growth factor receptors. In the case of melanoma in Xiphophorus fish, the oncogenic EGF receptor orthologue Xiphophorus melanoma receptor kinase (Xmrk) effects continuous activation of the Src family kinase Fyn, but not of the other family members Src or Yes. Here, Fyn is strongly involved in promoting many tumorigenic events. Although Fyn is expressed in most mammalian tissues, there are only few reports of its involvement in the development of solid tumors. To find out whether the prominent role of Xiphophorus Fyn is based on an altered binding to its important binding partner FAK when compared to its mammalian Fyn counterparts, we performed yeast-two-hybrid analyses. We compared Xiphophorus and murine Fyn with respect to their binding to full-length and truncated FAK constructs. We found that interaction with FAK occurs similarly for Xiphophorus and mouse Fyn. Both phosphorylated FAK residue Y397 and FAK proline-rich domain are involved in Fyn binding. We also found interaction of FAK and Fyn in human melanoma cell lines. These data suggest a possible, yet unrecognized role of Fyn in the tumorigenesis of human melanoma, too.     

Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.

The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.     

DNA repair in hybrid fish of the genus Xiphophorus

The genus Xiphophorus is an important vertebrate model for investigating the etiology and genetics of both spontaneous and induced cancers. Xiphophorus are comprised of 23 species most of which can be crossed to produce fertile interspecies hybrid progeny. The Xiphophorus gene map is well developed and allows genetic associations to be studied among cohorts of progeny derived from backcrossing interspecies hybrid animals to one of the parental strains. In interspecies cross-progeny from select Xiphophorus backcrosses, ionizing radiation, ultraviolet light (UVB), and exposure to methylnitrosourea (MNU) have all been shown to induce tumors. Induced tumor types represented in various models include melanoma, fibrosarcoma, schwannoma, retinoblastoma, etc. The well-established backcross hybrid genetics make Xiphophorus fish an excellent system to study the contribution of DNA repair capability to induced tumorigenesis. DNA repair pathways represent multigenic traits that must be tightly regulated to insure genome fidelity. Herein we review initial DNA repair studies that assess repair capacities among different Xiphophorus species and interspecies hybrids. Assessment of both base excision repair (BER) and nucleotide excision repair (NER) have yielded consistent results indicating reduced DNA repair function in hybrid fish tissues. These data provide molecular support for potential reduced fitness in hybrid fish under conditions of environmental stress and may present a plausible explanation for absence of interspecies hybridization in sympatric environments. In addition, they support the role of direct DNA damage and its repair in the initiation of tumors in Xiphophorus hybrids.     

Homology of Melanoma-Inducing Loci in the Genus Xiphophorus

Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Some of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern loci from the playfish Xiphophorus maculatus the melanoma-inducing gene has been cloned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanoma-predisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a gene-duplication event, thereby acquiring the oncogenic potential. The homology of the melanoma-inducing loci points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.     

Applications of mouse glioma models in preclinical trials

Gliomas are the most common primary tumors that arise from glial cells and their precursors in the central nervous system. Most of the genetic alterations identified in human gliomas result in signal transduction abnormalities or disruption of cell cycle arrest pathways. Over the past years, several mouse glioma models have been generated based on human genetic abnormalities and the induced gliomas exhibit histological similarities to their human counterparts. There is emerging evidence suggesting that an oncogenic signaling initiating tumorigenesis is also required for tumor maintenance, these glioma models can be used to further characterize the mechanisms of oncogenic signaling in tumor formation, as well as identify molecular targets in preclinical trials.     

The Xmrk receptor tyrosine kinase is activated in Xiphophorus malignant melanoma.

Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential ligand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properties and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related ligand-binding domain sequences of the human epidermal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk protein was examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transforming protein equivalent to other tyrosine kinase oncoproteins.     

Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sex-determining region of the platyfish Xiphophorus maculatus

Despite the major importance of sex determination in aquaculture, no master sex-determining gene has been identified so far in teleost fish. In the platyfish Xiphophorus maculatus, this master gene is flanked by two receptor tyrosine kinase genes, the Xmrk oncogene responsible for melanoma formation in some Xiphophorus interspecific hybrids, and its proto-oncogenic counterpart. Both Xmrk genes, which have already been characterised at the molecular level, delimit a region of about 1 Mb that contains other gene loci involved in sexual maturity, pigmentation and melanoma formation. We have constructed a genomic bacterial artificial chromosome (BAC) library of X. maculatus with a tenfold coverage of the haploid genome and walked on both X and Y sex chromosomes starting from both Xmrk genes. This led to the assembly of BAC contigs from the sex-determining region covering approximately 950 kb of the X and 750 kb of the Y chromosome. To our knowledge, these are the largest contigs reported so far for sex chromosomes in fish. Molecular analysis suggests that the sex-determining region of X. maculatus frequently undergoes retrotranspositions and other kinds of rearrangements. This genomic plasticity might be related to the high genetic variability observed in Xiphophorus for sex determination, sexual maturity, pigmentation and melanoma formation, which are encoded by gene loci located in the sex-determining region.     

The eye of Drosophila as a model system for studying intracellular signaling in ontogenesis and pathogenesis

Many human diseases are caused by malfunction of basic types of cellular activity such as proliferation, differentiation, apoptosis, cell polarization, and migration. In turn, these processes are associated with different routes of intracellular signal transduction. A number of model systems have been designed to study normal and abnormal cellular and molecular processes associated with pathogenesis. The developing eye of the fruit fly Drosophila melanogaster is one of these systems. The sequential development of compound eyes of this insect makes it possible to model human neurodegenerative diseases and mechanisms of carcinogenesis. In this paper we overview the program of the eye development in Drosophila, with emphasis on intracellular signaling pathways that regulate this complex process. We discuss in detail the roles of the Notch, Hedgehog, TGFβ, Wnt, and receptor tyrosine kinase signaling pathways in Drosophila eye development and human pathology. We also briefly describe the modern methods of experimentation with this model organism to analyze the function of human pathogenic proteins.     

PI3-Kinase Is Involved in Mitogenic Signaling by the Oncogenic Receptor Tyrosine Kinase Xiphophorus Melanoma Receptor Kinase in Fish Melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.     

The lipid raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.     

The Met receptor tyrosine kinase and basal breast cancer

Breast cancer is a complex disease that comprises cancers of distinct biologies and responses to treatment. Clinical management relies on traditional clinicopathological parameters, involving lymph node status, histological grade, as well as expression of the estrogen receptor or human epidermal growth factor receptor 2. Molecular pathology as well as protein and gene expression profiling have divided breast tumors into molecular subtypes associated with different clinical outcomes. One of these, defined as basal breast cancer, is associated with poor prognosis. Molecular mechanisms involved in the induction of basal breast cancer are poorly understood and targeted therapies for this subtype are lacking. Recent evidence using murine models identified a role for the Met receptor tyrosine kinase in the induction of murine mammary tumors with characteristics of human basal breast cancers. Moreover, elevated Met protein and RNA is associated with human basal tumors and poor outcome. These studies identify a link between the Met receptor tyrosine kinase, epithelial mesenchymal transition, and basal breast cancer. In this review, we provide an overview of murine Met models in relation to the spectrum of mouse models of breast cancer and a role for the Met receptor in basal breast cancer tumorigenesis.     

c-Kit 激酶抑制剂的研究与开发

c-Kit 是典型的Ⅲ型受体酪氨酸激酶,在肿瘤的发生发展以及侵袭、迁移和复发过程中起着十分重要的作用,是目前肿瘤分子靶向治疗的热门靶标之一,其抑制剂也成为抗肿瘤药物研究与开发的热点。简介c-Kit 激酶及其激活型突变与肿瘤发生发展的关系,着重综述近年来已上市和处于临床试验阶段的c-Kit 激酶抑制剂及其耐药机制研究。     

Kaposi's sarcoma-associated herpesvirus-induced upregulation of the c-kit proto-oncogene,as identified by gene expression profiling,is essential for the transformation of endothelial cells

Kaposi's sarcoma (KS), the most frequent malignancy afflicting AIDS patients, is characterized by spindle cell formation and vascularization. Infection with KS-associated herpesvirus (KSHV) is consistently observed in all forms of KS. Spindle cell formation can be replicated in vitro by infection of dermal microvascular endothelial cells (DMVEC) with KSHV. To study the molecular mechanism of this transformation, we compared RNA expression profiles of KSHV-infected and mock-infected DMVEC. Induction of several proto-oncogenes was observed, particularly the receptor tyrosine kinase c-kit. Consistent with increased c-Kit expression, KHSV-infected DMVEC displayed enhanced proliferation in response to the c-Kit ligand, stem cell factor (SCF). Inhibition of c-Kit activity with either a pharmacological inhibitor of c-Kit (STI 571) or a dominant-negative c-Kit protein reversed SCF-dependent proliferation. Importantly, inhibition of c-Kit signal transduction reversed the KSHV-induced morphological transformation of DMVEC. Furthermore, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation. Together, these data demonstrate an essential role for c-Kit in KS tumorigenesis and reveal a target for pharmacological intervention.     

Yeast as a model for studying human neurodegenerative disorders

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.     

The role of protein tyrosine phosphatases in colorectal cancer

Colorectal cancer is one of the most common oncogenic diseases in the Western world. Several cancer associated cellular pathways have been identified, in which protein phosphorylation and dephosphorylation, especially on tyrosine residues, are one of most abundant regulatory mechanisms. The balance between these processes is under tight control by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Aberrant activity of oncogenic PTKs is present in a large portion of human cancers. Because of the counteracting role of PTPs on phosphorylation-based activation of signal pathways, it has long been thought that PTPs must act as tumor suppressors. This dogma is now being challenged, with recent evidence showing that dephosphorylation events induced by some PTPs may actually stimulate tumor formation. As such, PTPs might form a novel attractive target for anticancer therapy. In this review, we summarize the action of different PTPs, the consequences of their altered expression in colorectal cancer, and their potential as target for the treatment of this deadly disease.