In mammals, Ins(1,4,5)P3, the well known calcium mobilization messenger, is phosphorylated in the cytosol at the 3-position of the inositol ring to yield Ins(1,3,4,5)P4 by Ins(1,4,5)P3 3-kinases A, B and C isoforms as well as by inositol polyphosphate multikinase (Ipmk). Studies in gene-deficient mice have revealed that these enzymes and Ins(1,3,4,5)P4, their reaction product, play essential role in multiple physiological processes, ranging from synaptic plasticity, hematopoietic cell survival, development and function, to mRNA export, transcriptional regulation and chromatin remodelling. Rather than to provide an unique and “universal” mechanism of Ins(1,3,4,5)P4 action, these studies in genetically-modified mice point for a role of this inositide in the control of calcium mobilization, of the subcellular localisation of PH domain-containing target proteins, and of higher inositol phosphate production. Mice deficient for the B isoform of inositol 1,4,5-trisphosphate 3-kinase (Itpkb) develop profound alterations in T and B cells as well as in neutrophils and mast cells. Our recent studies indicate that the 3-kinase Itpkb and Ins(1,3,4,5)P4 are important for the survival of naïve mature B cells and the control of proapoptotic Bim protein expression, rather than for the control of B cell transition from one developmental stage to another. They also suggest that Itpkb is an important component in the control of B cell anergy.
We have summarized here the importance of ChoKα1 in human carcinogenesis. ChoKα1 displays its oncogenic activity through activation of specific signaling pathways that influence on cell proliferation and survival. It is overexpressed in a large number of human tumors with an incidence of 40-60% of all tumors investigated. Currently, there is an active effort in the development of strategies to knockdown the activity of ChoKα through specific siRNA or small molecules inhibitors. Results from genetic silencing or from treatment with MN58b, a well characterized ChoKα inhibitor showing antiproliferative and antitumoral effect in mice xenografts, provide strong support to this concept, indicating that the design of new antitumoral drugs must be selective against this isoform. However, affecting the other two known isoforms of ChoK may have also therapeutic consequences since the physiologically active form of ChoK may be constituted by homo or heterodimers. Furthermore, alteration of the ChoKβ activity might lead to a change in the lipid content of the cells of particular tissues such as skeletal muscle as described in the ChoKβ null mice (Sher et al., 2006). Finally, the identification of the ChoKα1 isoform as an excellent novel tool for the diagnosis and prognosis of cancer patients may have clinical consequences of immediate usefulness. On one hand, the use of specific monoclonal antibodies against ChoKα1 as a tool for diagnosis in paraffin embedded samples from patient biopsies, through standard immunohistochemistry techniques, can now be achieved (Gallego-Ortega et al., 2006). On the other hand, it has been recently described the prognostic value of determination of ChoKα1 expression levels in non-small cell lung cancer using real time quantitative PCR technology (Ramírez de Molina et al., 2007). Therefore, further research should be supported on the utility of ChoK isoforms as a promising area to improve cancer diagnosis and treatment.
Ins and Ins phospholipids are present in and are made by most Archaea and all eukaryotes. Relatively few bacteria possess Ins phospholipids: and only one major grouping, the Actinobacteria, is known to have evolved multiple functions for Ins derivatives. The Ins phospholipids of all organisms, whether they have diradylglycerol or ceramide backbones, seem to use the same Ins1P headgroup stereochemistry, so they are probably made by evolutionarily conserved pathways. It seems likely that an early member of the Archaea made the first phospholipid with an Ins1P headgroup -maybe three billionyears ago – and that amuchlater archaeal descendentwas the ancestral contributor that brought these molecules into the common ancestor of all eukaryotes – maybe two billionyears ago (Michell, 2007, 2008). It will only be possible to infer the likely details of these processes when we have learned much more about the Ins lipid biochemistry of modern archaeons. All eukaryotes make substantial amounts of PtdIns, both as a ‘bulk’ membrane phospholipid and as the precursor of seven phosphorylated derivatives of PtdIns (the polyphosphoinositides; PPIn) and of the ‘GPI anchors’ of cell surface ectoproteins. PtdIns(4,5)P2 – with its many functions – and its precursor PtdIns4P are found in all in eukaryotes. So are PtdIns3P and PtdIns(3,5)P2, which have ubiquitous roles in the regulation of membrane trafficking events. However, synthesis of and signalling by PtdIns(3,4,5) P 3 appears to be confined to a later-evolved group of eukaryotes.
Mammals have at least two receptors for LTB4; high-affinity BLT1 and low-affinity BLT2, both of which are GPCRs. 12-HHT serves as a more potent and abundant ligand for BLT2 than LTB4. BLT1 is expressed in a variety of inflammatory and immune cells including granulocytes, eosinophils, macrophages, differentiated Th1, Th2 and Th17 cells, effecter CD8+ T cells, dendritic cells and osteoclasts. BLT1 antagonists will be beneficial for the treatment of various diseases such as bronchial asthma, multiple sclerosis, contact dermatitis, and postmenopausal osteoporosis. BLT2 plays different roles from BLT1, and one important role of BLT2 is the maintenance of mucosal integrity in the colon.
Cancer is a complex and heterogeneous disease marked by the dysregulation of cancer driver genes historically classified as oncogenes or tumour suppressors according to their ability to promote or inhibit tumour development and growth, respectively. Certain genes display both oncogenic and tumour suppressor functions depending on the biological context, and as such have been termed dual-role cancer driver genes. However, because of their context-dependent behaviour, the tumourigenic mechanism of many dual-role genes is elusive and remains a significant knowledge gap in our effort to understand and treat cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) is an emerging dual-role cancer driver gene, primarily known for its role as a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway.In response to growth factor stimulation, class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane. PtdIns(3,4,5)P3 can be hydrolysed by inositol polyphosphate 5-phosphatases to generate PtdIns(3,4)P2, which, together with PtdIns(3,4,5)P3, facilitates the activation of AKT to promote cell proliferation, survival, migration, and metabolism. Phosphatase and tensin homology on chromosome 10 (PTEN) and INPP4B are dual-specificity phosphatases that hydrolyse PtdIns(3,4,5)P3 and PtdIns(3,4)P2, respectively, and thus negatively regulate PI3K/AKT signalling. PTEN is a bona fide tumour suppressor that is frequently lost in human tumours. INPP4B was initially characterised as a tumour suppressor akin to PTEN, and has been implicated as such in a number of cancers, including prostate, thyroid, and basal-like breast cancers. However, evidence has since emerged revealing INPP4B as a paradoxical oncogene in several malignancies, with increased INPP4B expression reported in AML, melanoma and colon cancers among others. Although the tumour suppressive function of INPP4B has been mostly ascribed to its ability to negatively regulate PI3K/AKT signalling, its oncogenic function remains less clear, with proposed mechanisms including promotion of PtdIns(3)P-dependent SGK3 signalling, inhibition of PTEN-dependent AKT activation, and enhancing DNA repair mechanisms to confer chemoresistance. Nevertheless, research is ongoing to identify the factors that dictate the tumourigenic output of INPP4B in different human cancers.In this review we discuss the dualistic role that INPP4B plays in the context of cancer development, progression and treatment, drawing comparisons to PTEN to explore how their similarities and, importantly, their differences may account for their diverging roles in tumourigenesis.
Transcriptome analysis of cancer cells has developed into a standard procedure to elucidate multiple features of the malignant process and to link gene expression to clinical properties. Gene expression profiling based on microarrays provides essentially correlative information and needs to be transferred to the functional level in order to understand the activity and contribution of individual genes or sets of genes as elements of the gene signature. To date, there exist significant gaps in the functional understanding of gene expression profiles. Moreover, the processes that drive the profound transcriptional alterations that characterize cancer cells remain mainly elusive. We have used pathway-restricted gene expression profiles derived from RAS oncogene-transformed cells and from RAS-expressing cancer cells to identify regulators downstream of the MAPK pathway.We describe the role of epigenetic regulation exemplified by the control of several immune genes in generic cell lines and colorectal cancer cells, particularly the functional interaction between signaling and DNA methylation. Moreover, we assess the role of the architectural transcription factor high mobility AT-hook 2 (HMGA2) as a regulator of the RAS-responsive transcriptome in ovarian epithelial cells. Finally, we describe an integrated approach combining pathway interference in colorectal cancer cells, gene expression profiling and computational analysis of regulatory elements of deregulated target genes. This strategy resulted in the identification of Y-box binding protein 1 (YBX1) as a regulator of MAPK-dependent proliferation and gene expression. The implications for a therapeutic application of HMGA2 gene silencing and the role of YBX1 as a prognostic factor are discussed.
Integrin linked kinase (ILK) is ubiquitously expressed serine/threonine protein kinase, a binding partner of β1 and β3 integrin subunit as a cytoplasmic effector of integrin receptors that functionally links them to the actin cytoskeleton.We postulate that ILK is important enzyme involved in epithelial-mesenchymal transition (EMT) a critical event in the process of cancer progression. Commonly used EMT molecular markers include among others increased expression of N-cadherin and vimentin, nuclear localization of β-catenin, and the decrease of E-cadherin synthesis. In this study we were able to show that N-cadherin expression in melanoma cells is dependent on ILK signaling and the translocation of β-catenin to the nucleus. Silencing of ILK expression by siRNA significantly inhibited the stabilization and subsequent nuclear translocation of β-catenin and the expression of N-cadherin, a crucial molecule in the EMT, which facilitates association with fibroblast and endothelial cells during invasion of various cancers. The results allow to cautiously speculate on the important role of ILK in the cross-talk between integrins and cadherins accompanying EMT in melanoma.
We have isolated cell with the cancer initiating cell (CIC) phenotype from PC3 cells. The PC3/(CIC) cells are more resistant than the PC3/(BC) cells to chemotherapeutic drugs such as docetaxel which is used to treat prostate cancer. Thus these prostate CICs could lay dormant and persist even after chemotherapeutic drug treatment. Then when the chemotherapeutic drug is removed, they could potentially repopulate the original tumor site or metastize to a distant site. However, the prostate CICs were not significantly more resistant to drugs which target EGFR, NF-κB, Smo and the natural product genistein. Interesting the prostate CICs could be rendered more sensitive to docetaxel by inclusion of suboptimal doses of genistein, cyclopamine, and EGFR inhibitors. In contrast, addition of suboptimal amounts of genistein, cyclopamine, or EGFR inhibitors did not increase the sensitivity of the PC/(BC) cells to docetaxel. Similar results were observed when combination experiments were performed with cyclopamine and suboptimal doses of either genistein or docetaxel. The BC cells are usually more rapidly proliferating than the CICs. Thus the CICs are not as sensitive to docetaxel which targets replication. In contrast, the CICs could be rendered sensitive to docetaxel or cyclopamine by co-treatment with certain other drugs, including the natural product genistein which is present in the human diet of many people, especially Asians. Genistein is by itself only weakly toxic to prostate and other cancer cells. That is probably one of the big reasons that it can be used as a dietary supplement for prostate and breast cancers. It is clear from our studies that low doses of genistein can increase the sensitivity of prostate CICs to drugs such as docetaxel and cyclopamine, two drugs either used or under consideration for prostate cancer therapy.
Gene loops are dynamic structures that juxtapose promoter–terminator regions of Pol II-transcribed genes. Although first described in yeast, gene loops have now been identified in yeast and mammalian cells. Looping requires components of the transcription preinitiation complex, the pre-mRNA 30-end processing machinery, and subunits of the nuclear pore complex. Loop formation is transcription-dependent, but neither basal nor activated transcription requires looping. Rather, looping appears to affect cellular memory of recent transcriptional activity, enabling a more rapid response to subsequent stimuli. The nuclear pore has been implicated in both memory and looping. Our working model is that loops are formed and/or maintained at the nuclear pore to facilitate hand-off of Pol II form the terminator to the promoter, thereby bypassing Pol II recruitment as the rate-limiting step in reactivation of transcription. Involvement of the nuclear pore also suggests that looping might facilitate mRNA export to the cytoplasm. The technology now exists to test these ideas.
The SREBP family of transcription factors regulates the expression of genes involved in fatty acid and cholesterol biosynthesis. The activation of SREBP transcription factors requires proteolytic cleavage of the inactive precursor and nuclear translocation of the mature form of the protein. It has been shown that nuclear accumulation of the mature form of SREBP1 is induced in response to activation of the serine/threonine kinase Akt, an important effector of the Ras/PI3-kinase signalling pathway. Activation of SREBP by Akt depends on the mammalian target of rapamycin complex 1 (mTORC1) but the exact mechanism of this activation remains unclear. We have investigated whether ablation of different signalling molecules downstream of mTORC1 affects expression of SREBP targets genes. We could show that inhibition of S6-kinases 1 and 2 expression using RNA interference did not block induction of expression of fatty acid synthase (FASN) or ATP-citrate lyase (ACLY) following activation of Akt in human retinal pigment epithelial cells. Furthermore, accumulation of mature SREBP1 was not inhibited after combined silencing of S6-kinases 1 and 2. Genetic ablation of both kinases also did not prevent the formation of mature SREBP1 in mouse embryonic fibroblasts. Taken together, these results suggest that S6-kinases 1 and 2 are dispensable for the induction of SREBP processing in the experimental systems used here.
The eukaryotic BBSome is a transport complex within cilia and assembled by chaperonin-like BBS proteins. Recent work indicates nuclear functions for BBS proteins in mammals, but it is unclear how common these are in extant proteins or when they evolved. We screened for BBS orthologues across a diverse set of eukaryotes, consolidated nuclear association via signal sequence predictions and permutation analysis, and validated nuclear localization in mammalian cells via fractionation and immunocytochemistry. BBS proteins are—with exceptions—conserved as a set in ciliated species. Predictions highlight five most likely nuclear proteins and suggest that nuclear roles evolved independently of nuclear access during mitosis. Nuclear localization was confirmed in human cells. These findings suggest that nuclear BBS functions are potentially not restricted to mammals, but may be a common frequently co-opted eukaryotic feature. Understanding the functional spectrum of BBS proteins will help elucidating their role in gene regulation, development, and disease.
These findings emphasize that the mTOR pathway may contribute to maintenance of quiescence of CSCs, and provide a basis for manipulating CSCs in the treatment of GBM. Future research should focus on further defining the PI3K/Akt/mTOR molecular network in the regulation of stem cell quiescence and provide rationale for targeting the cancer-initiating cells of GBM.
Neutral sphingomyelinases (N-SMases) are considered to be key mediators of stress-induced ceramide production. The extended family of N-SMases is a subset of the DNaseI superfamily and comprises members from bacteria, yeast and mammals. In recent years, the identification and cloning of mammalian N-SMase family members has led to significant advances in understanding their physiological roles and regulation. However, there is still limited information on their regulation at the biochemical and molecular level. In this review, we summarize current knowledge about the biochemical regulation of the eukaryotic N-SMases and identify the major areas where knowledge is lacking. In recent years, research into the roles and regulation of N-SMases has moved in great strides with the cloning and characterization of multiple N-SMase isoforms and the development of knockout mice. However, as researchers continue to move forward in understanding the physiological functions of these various N-SMase isoforms, it has become exceedingly important to define howthese isoforms are regulated at the biochemical and molecular level. This is crucial for the development of future tools to study N-SMase signaling such as, for example, phospho-specific antibodies designating activation states. This is also an important part of identifying novel roles of N-SMases in physiological and pathological states. Finally, only by obtaining a more complete understanding of the workings of these enzymes at the molecular level, will investigators be able to design appropriate compounds that can target and inhibit their activity both efficiently and specifically. Certainly, the last of these is crucial when considering the potential of N-SMases as therapeutic targets. With this in mind, we sincerely hope that the next decade of research will even surpass the last ten years in advancing our understanding of the eukaryotic N-SMase family.
PI3Ks catalyze the phosphorylation of the inositol hydroxyls of phosphoinositide membrane components. The changes in phosphorylation of the inositides recruit proteins to the plasma membrane that initiate important signaling cascades. PI3Kα, one of the class IA PI3Ks, is highly mutated in cancers. All mutations analyzed result in an increase in enzymatic activity. The structures of this enzyme determined by X-ray diffraction, provide a framework for analyzing the possible structural effect of these mutations and their effect on the enzymatic activity. Many of the mutations occur at domain interfaces where they can affect domain interactions and relieve the inhibition of the wild-type enzyme by the nSH2 domain of p85. This mechanism is analogous to the mechanism of physiological activation by activated tyrosine-kinase receptors in which the phosphorylated tyrosine of the receptor (or their substrates) dislodges the nSH2 from its inhibitory position in the complex by competing with its binding to a loop in the helical domain. Other mutations in the kinase domain can directly affect the conformation of the catalytic site. One mutation, His1047Arg, uses a completely different mechanism: it changes the conformation of the C-terminal loop in such a way that it increases the interaction of the enzyme with the membrane, granting increased access to the phosphoinositide substrates. Taking advantage of the reliance of some cancers on the increased activity of mutated PI3Kα, will require the development of isoform-specific, mutant-specific inhibitors. The structural, biochemical and physiological data that are becoming available for PI3Ks are an important first step in this direction.
Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.
The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCβ1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCβ1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCβ1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCβ1 expression in MDS has been reviewed as well.
Gene alterations affecting elements of PI3K signaling pathway do not appear to be sufficient to explain the extremely high frequency of PI3K signaling hyperactivation in leukemia. It has been known for long that PTEN phosphorylation at the C-terminal tail, in particular by CK2, contributes to the stabilization and simultaneous inhibition of this critical tumor suppressor. However, direct evidence of the involvement of this mechanism in cancer has been gathered only recently. It is now known that CK2-mediated posttranslational, non-deleting, inactivation of PTEN occurs in T-ALL, CLL and probably other leukemias and solid tumors. To explore this knowledge for therapeutic purposes remains one of the challenges ahead.
Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.
The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access.
