Genotranscriptomic meta-analysis of the Polycomb gene CBX2 in human cancers: initial evidence of an oncogenic role.

BACKGROUND: Polycomb group (PcG) proteins are histone modifiers known to transcriptionally silence key tumour suppressor genes in multiple human cancers. The chromobox proteins (CBX2, 4, 6, 7, and 8) are critical components of PcG-mediated repression. Four of them have been associated with tumour biology, but the role of CBX2 in cancer remains largely uncharacterised. METHODS: Addressing this issue, we conducted a comprehensive and unbiased genotranscriptomic meta-analysis of CBX2 in human cancers using the COSMIC and Oncomine databases. RESULTS: We discovered changes in gene expression that are suggestive of a widespread oncogenic role for CBX2. Our genetic analysis of 8013 tumours spanning 29 tissue types revealed no inactivating chromosomal aberrations and only 40 point mutations at the CBX2 locus. In contrast, the overall rate of CBX2 amplification averaged 10% in all combined neoplasms but exceeded 30% in ovarian, breast, and lung tumours. In addition, transcriptomic analyses revealed a strong tendency for increased CBX2 mRNA levels in many cancers compared with normal tissues, independently of CDKN2A/B silencing. Furthermore, CBX2 upregulation and amplification significantly correlated with metastatic progression and lower overall survival in many cancer types, particularly those of the breast. CONCLUSIONS: Overall, we report that the molecular profile of CBX2 is suggestive of an oncogenic role. As CBX2 has never been studied in human neoplasms, our results provide the rationale to further investigate the function of CBX2 in the context of cancer cells.

Mutational analysis of Polycomb genes in solid tumours identifies PHC3 amplification as a possible cancer-driving genetic alteration.

BACKGROUND: Polycomb group genes (PcGs) are epigenetic effectors implicated in most cancer hallmarks. The mutational status of all PcGs has never been systematically assessed in solid tumours. METHODS: We conducted a multi-step analysis on publically available databases and patient samples to identify somatic aberrations of PcGs. RESULTS: Data from more than 1000 cancer patients show for the first time that the PcG member PHC3 is amplified in three epithelial neoplasms (rate: 8-35%). This aberration predicts poorer prognosis in lung and uterine carcinomas (P<0.01). Gene amplification correlates with mRNA overexpression (P<0.01), suggesting a functional role of this aberration. CONCLUSION: PHC3 amplification may emerge as a biomarker and potential therapeutic target in a relevant fraction of epithelial tumours.

Paracrine signalling loops in adult human and mouse pancreatic islets: netrins modulate beta cell apoptosis signalling via dependence receptors.

AIMS/HYPOTHESIS: Adult pancreatic islets contain multiple cell types that produce and secrete well characterised hormones, including insulin, glucagon and somatostatin. Although it is increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. We therefore sought to identify potential autocrine and/or paracrine islet growth factor loops, and to characterise the function of the netrin family of islet-secreted factors and their receptors, which have been previously unreported in adult islets. METHODS: Gene expression databases, islet-specific tag sequencing libraries and microarray datasets of FACS purified beta cells were used to compile a list of secreted factors and receptors present in mouse or human islets. Netrins and their receptors were further assessed using RT-PCR, Western blot analysis and immunofluorescence staining. The roles of netrin-1 and netrin-4 in beta cell function, apoptosis and proliferation were also examined. RESULTS: We identified 233 secreted factors and 234 secreted factor receptors in islets. The presence of netrins and their receptors was further confirmed. Downregulation of caspase-3 activation was observed when MIN6 cells were exposed to exogenous netrin-1 and netrin-4 under hyperglycaemic conditions. Reduction in caspase-3 cleavage was linked to the decrease in dependence receptors, neogenin and unc-5 homologue A, as well as the activation of Akt and extracellular signal-regulated protein kinase (ERK) signalling. CONCLUSIONS/INTERPRETATION: Our results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in adult beta cells. Since diabetes results from a deficiency in functional beta cell mass, these studies are important steps towards developing novel therapies to improve beta cell survival.

Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice.

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1(tm1Bc) to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1(tm1Bc) mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.

Identification of novel genes and transcription factors involved in spleen, thymus and immunological development and function.

We constructed and analyzed six serial analysis of gene expression (SAGE) libraries to identify genes with previously uncharacterized roles in spleen or thymus development. A total of 625 070 tags were sequenced from the three spleen (embryonic day (E)15.5, E16.5 and adult) and three thymus (E15.5, E18.5 and adult) libraries. These tags corresponded to 83 182 tag types, which mapped unambiguously to 36 133 different genes. Genes over-represented in these libraries, compared to 115 mouse SAGE libraries (www.mouseatlas.org), included genes of known and unknown immunological or developmental relevance. The expression profiles of 11 genes with unknown roles in spleen and thymus development were validated using reverse transcription-qPCR. We further characterized the expression of one of these candidates, RIKEN cDNA 9230105E10 that encodes a murine homolog of Trim5alpha, in numerous adult tissues and immune cell types. In addition, we demonstrate that transcript levels are upregulated in response to TLR stimulation of plasmacytoid dendritic cells and macrophages. This work provides the first evidence of regulated and cell type-specific expression of this gene. In addition, these observations suggest that the SAGE libraries provide an important resource for further investigations into the molecular mechanisms regulating spleen and thymus organogenesis, as well as the development of immunological competence.

Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product.

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain--mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain--containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase--activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease.

Partially distinct molecular mechanisms mediate inhibitory FcgammaRIIB signaling in resting and activated B cells.

FcgammaRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for FcgammaRIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient FcgammaRIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, FcgammaRIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-2. Finally, data suggest that both SHIP-2 and SHIP can mediate downstream biologic consequences of FcgammaRIIB signaling, including inhibition of the proliferative response.

Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.

Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp(-/-) mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp(-/-) mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp(-/-) mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.

A regulatory role for Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) in phagocytosis mediated by Fc gamma receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18).

The Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) is recruited to immunoreceptor tyrosine-based inhibition motif (ITIM)-containing proteins, thereby suppressing phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. The role of SHIP in phagocytosis, a PI 3-kinase-dependent pathway, is unknown. Overexpression of SHIP in macrophages led to an inhibition of phagocytosis mediated by receptors for the Fc portion of IgG (Fc gamma Rs). In contrast, macrophages expressing catalytically inactive SHIP or lacking SHIP expression demonstrated enhanced phagocytosis. To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; alpha(M)beta(2))-dependent phagocytosis. Macrophages overexpressing SHIP demonstrated impaired CR3-mediated phagocytosis, whereas macrophages expressing catalytically inactive SHIP demonstrated enhanced phagocytosis. CR3-mediated phagocytosis in macrophages derived from SHIP(-/-) mice was up to 2.5 times as efficient as that observed in macrophages derived from littermate controls. SHIP was localized to Fc gamma R- and CR3-containing phagocytic cups and was recruited to the cytoskeleton upon clustering of CR3. In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis. We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating beta(2) integrin outside-in signaling.

A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase.

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5' inositol phosphatase (SHIP).

Although the Src homology 2 domain-containing 5' inositol phosphatase (SHIP) is a well-known mediator of inhibitory signals after B cell antigen receptor (BCR) coaggregation with the low affinity Fc receptor, it is not known whether SHIP functions to inhibit signals after stimulation through the BCR alone. Here, we show using gene-ablated mice that SHIP is a crucial regulator of BCR-mediated signaling, B cell activation, and B cell development. We demonstrate a critical role for SHIP in termination of phosphatidylinositol 3,4,5-triphosphate (PI[3,4,5]P(3)) signals that follow BCR aggregation. Consistent with enhanced PI(3,4,5)P(3) signaling, we find that splenic B cells from SHIP-deficient mice display enhanced sensitivity to BCR-mediated induction of the activation markers CD86 and CD69. We further demonstrate that SHIP regulates the rate of B cell development in the bone marrow and spleen, as B cell precursors from SHIP-deficient mice progress more rapidly through the immature and transitional developmental stages. Finally, we observe that SHIP-deficient B cells have increased resistance to BCR-mediated cell death. These results demonstrate a central role for SHIP in regulation of BCR signaling and B cell biology, from signal driven development in the bone marrow and spleen, to activation and death in the periphery.

A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice.

In this report, we demonstrate that the Src homology 2 domain-containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP(-/-) mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP(-/)- B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcgamma receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP(-/)- mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell-independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.

Huntingtin is required for normal hematopoiesis.

Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.

The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells.

The low affinity receptor for IgG, FcgammaRIIB, functions to dampen the antibody response and reduce the risk of autoimmunity. This function is reportedly mediated in part by inhibition of B cell antigen receptor (BCR)-mediated p21ras activation, though the basis of this inhibition is unknown. We show here that FcgammaRIIB-BCR coaggregation leads to increased tyrosine phosphorylation of the RasGAP-binding protein p62dok, with a concomitant increase in its binding to RasGAP. These effects require the recruitment and tyrosine phosphorylation of the phosphatidylinositol 5-phosphatase SHIP, which further recruits p62dok via the latter's phosphotyrosine-binding domain. Using chimeric FcgammaRIIB containing the RasGAP-binding domain of p62dok, we demonstrate that p62dok contains all structural information required to mediate the inhibitory effect of FcgammaRIIB on Erk activation.

Altered responsiveness to chemokines due to targeted disruption of SHIP.

SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase- (PI-3K-) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell-derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP(-/-) cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.

SHIPs ahoy.

In 1996 three groups independently cloned a hemopoietic specific, src homology 2-containing inositol 5'-phosphatase which, based on its structure, was called SHIP. More recently, a second more widely expressed SHIP-like protein has been cloned and called SHIP2. Both specifically hydrolyze phosphatidylinositol-3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vitro. Moreover, SHIP has been shown in vivo to be the primary enzyme responsible for breaking down phosphatidylinositol-3,4,5-trisphosphate to phosphatidylinositol-3,4-bisphosphate in normal mast cells and, as a result, limits normal and prevents inappropriate mast cell degranulation. Because of their ability to break down phosphatidylinositol-3,4,5-trisphosphate, the SHIPs have the potential to regulate many, if not all, phosphatidylinositol-3-kinase induced events including, proliferation, differentiation, apoptosis, end cell activation, cell movement and adhesion and will thus likely be the subject of intensive research over the next few years.

The role of SHIP in growth factor induced signalling.

The recently cloned, hemopoietic-specific, src homology 2 (SH2)-containing inositol phosphatase, SHIP, is rapidly gaining prominence as a potential regulator of all phosphatidylinositol (PI)-3 kinase mediated events since it has been shown both in vitro and in vivo to hydrolyze the 5' phosphate from phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3). Thus SHIP, and its more widely expressed counterpart, SHIP2, could play a central role in determining PI-3,4,5-P3 and PI-3,4-P2 levels in many cell types. To explore the in vivo function of SHIP further we recently generated a SHIP knock out mouse and in this review we discuss experiments carried out with bone marrow derived mast cells (BMMCs) from these animals.

Life without huntingtin: normal differentiation into functional neurons.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells.

To investigate the role of the src homology 2 (SH2)-containing inositol 5' phosphatase (SHIP) in growth factor-mediated signalling, we compared Steel factor (SF)-induced events in bone marrow-derived mast cells (BMMCs) from SHIP-/- and SHIP+/+ littermates. We found SF alone stimulated massive degranulation from SHIP-/- but none from SHIP+/+ BMMCs. This SF-induced degranulation, which was not due to higher c-kit levels in SHIP-/- cells, correlated with higher intracellular calcium than that in SHIP+/+ cells and was dependent on the influx of extracellular calcium. Both this influx and subsequent degranulation were completely inhibited by PI-3-kinase inhibitors, indicating that SF-induced activation of PI-3-kinase was upstream of extracellular calcium entry. A comparison of phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels following SF stimulation of SHIP+/+ and SHIP-/- BMMCs suggested that SHIP restricted this entry by hydrolyzing PIP3. Although PI-3-kinase inhibitors blocked the release of intracellular calcium, implicating PIP3, and PLCgamma-2 was slightly more tyrosine phosphorylated in SHIP-/- cells, the increase in inositol-1,4,5-trisphosphate (IP3) and intracellular calcium levels were identical in SHIP-/- and SHIP+/+ BMMCs. These results suggest that SHIP prevents SF from triggering degranulation of normal BMMCs, and does so by hydrolyzing PIP3, which in turn limits extracellular calcium entry at a step after the release of intracellular calcium.