Fifteen rare cancer-derived mutants of PIK3CA, the gene coding for the catalytic subunit p110␣ of phosphatidylinositol 3-kinase (PI3K), were examined for their biological and biochemical properties. Fourteen of these mutants show a gain of function: they induce rapamycin-sensitive oncogenic transformation of chicken embryo fibroblasts, constitutively activate Akt and TOR-mediated signaling, and show enhanced lipid kinase activity. Mapping of these mutants on a partial structural model of p110␣ suggests three groups of mutants, defined by their location in distinct functional domains of the protein. We hypothesize that each of these three groups induces a gain of PI3K function by a different molecular mechanism. Mutants in the C2 domain increase the positive surface charge of this domain and therefore may enhance the recruitment of p110␣ to cellular membranes. Mutants in the helical domain map to a contiguous surface of the protein and may affect the interaction with other protein(s). Mutants in the kinase domain are located near the hinge of the activation loop. They may alter the position and mobility of the activation loop. Arbitrarily introduced mutations that have no detectable phenotype map either to the interior of the protein or are positioned on a surface region that lies opposite to the exposed surfaces containing gain-of-function mutants. Engineered mutants that exchange acidic or neutral residues for basic residues on the critical surfaces show a gain of function. molecular model ͉ phosphatidylinositol 3-kinase ͉ lipid membrane ͉ protein-protein interaction ͉ activation loop T he catalytic subunit p110␣ of class I phosphatidylinositol 3-kinase (PI3K) is frequently mutated in cancer (1-5). The incidence of these cancer-specific point mutations varies with the tissue of origin and is particularly high in mammary and colorectal cancers (1, 2, 6 -12). A conspicuous feature of the p110␣ mutations is their specific location in the gene of p110␣: almost 80% of these mutations map to one of three hot spots in the p110␣ coding sequence (1). Two of the hot spot mutations are located in the helical domain of p110␣ at residue positions E542K and E545K, and the third hot spot (residue H1047R) is located in the C-terminal portion of the kinase domain. Besides these frequently occurring hot spot mutations, numerous rare, cancer-specific mutations have also been identified in p110␣ (1,3,7,8,10,11). These rare mutations are widely distributed over the entire coding sequence of p110␣. It has been assumed that the prevalence of hot spot mutations ref lects the strong selective growth advantage that these mutants provide to the cell. This suggestion is supported by the fact that all three hot spot mutations confer in vitro and in vivo oncogenicity on p110␣ (13-18). A corollary of this interpretation is that the rare mutants would not offer a comparable growth advantage and may therefore show either no phenotype or a loss of function. Consequently, they may be of no relevance to the oncogenic process. We studied 15 of the...
Ether-á-go-go-1 (Eag1) is a CNS-localized voltage-gated potassium channel that is found ectopically expressed in a majority of extracranial solid tumors. While circumstantial evidence linking Eag1 to tumor biology has been well established, the mechanisms by which the channel contributes to tumor progression remain elusive. In this study, we have used in vivo and in vitro techniques to identify a candidate mechanism. A mutation that eliminates ion permeation fails to completely abolish xenograft tumor formation by transfected cells, indicating that Eag1 contributes to tumor progression independently of its primary function as an ion channel. Our data suggest that Eag1 interferes with the cellular mechanism for maintaining oxygen homeostasis, increasing HIF-1 activity, and thereby VEGF secretion and tumor vascularization.
BackgroundPrimary and secondary brain cancers are highly treatment resistant, and their marked angiogenesis attracts interest as a potential therapeutic target. Recent observations reveal that the microvascular endothelium of primary high-grade gliomas expresses prostate specific membrane antigen (PSMA). Breast cancers express PSMA and they frequently form secondary brain tumors. Hence we report here our pilot study addressing the feasibility of PSMA targeting in brain and metastatic breast tumors, by examining PSMA levels in all glioma grades (19 patients) and in breast cancer brain metastases (5 patients).MethodsTumor specimens were acquired from archival material and normal brain tissues from autopsies. Tissue were stained and probed for PSMA, and the expression levels imaged and quantified using automated hardware and software. PSMA staining intensities of glioma subtypes, breast tumors, and breast tumor brain metastases were compared statistically versus normals.ResultsNormal brain microvessels (4 autopsies) did not stain for PSMA, while a small proportion (<5%) of healthy neurons stained, and were surrounded by an intact blood brain barrier. Tumor microvessels of the highly angiogenic grade IV gliomas showed intense PSMA staining which varied between patients and was significantly higher (p < 0.05) than normal brain. Grade I gliomas showed moderate vessel staining, while grade II and III gliomas had no vessel staining, but a few (<2%) of the tumor cells stained. Both primary breast cancer tissues and the associated brain metastases exhibited vascular PSMA staining, although the intensity of staining was generally less for the metastatic lesions.ConclusionsOur results align with and extend previous data showing PSMA expression in blood vessels of gliomas and breast cancer brain metastases. These results provide a rationale for more comprehensive studies to explore PSMA targeted agents for treating secondary brain tumors with PSMA expressing vasculature. Moreover, given that PSMA participates in angiogenesis, cell signaling, tumor survival, and invasion, characterizing its expression may help guide later investigations of the poorly understood process of low grade glioma progression to glioblastoma.
Calcium transients play an important role in the early and later phases of differentiation and maturation of single neurons and neuronal networks. Small-conductance calcium-activated potassium channels of the SK type modulate membrane excitability and are important determinants of the firing properties of central neurons. Increases in the intracellular calcium concentration activate SK channels, leading to a hyperpolarization of the membrane potential, which in turn reduces the calcium inflow into the cell. This feedback mechanism is ideally suited to regulate the spatiotemporal occurrence of calcium transients. However, the role of SK channels in neuronal development has not been addressed so far. We have concentrated on the ontogenesis and function of SK channels in the developing rat cerebellum, focusing particularly on Purkinje neurons. Electrophysiological recordings combined with specific pharmacological tools have revealed for the first time the presence of an afterhyperpolarizing current (I(AHP)) in immature Purkinje cells in rat cerebellar slices. The channel subunits underlying this current were identified as SK2 and localized by in situ hybridization and subunit-specific antibodies. Their expression level was shown to be high at birth and subsequently to decline during the first 3 weeks of postnatal life, both at the mRNA and protein levels. This developmental regulation was tightly correlated with the expression of I(AHP) and the prominent role of SK2 channels in shaping the spontaneous firing pattern in young, but not in adult, Purkinje neurons. These results provide the first evidence of the developmental regulation and function of SK channels in central neurons.
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