Heterochromatin protein 1 expression is reduced in human thyroid malignancy.

Owing to the loss of heterochromatin integrity that occurs during thyroid tumorigenesis, the expression of Heterochromatin Protein 1 isoforms HP1α and HP1ß was assessed by immunohistochemistry in 189 thyroid tumors and non-neoplastic tissues. Expression of HP1ß was significantly decreased in all thyroid lesions, except in follicular adenomas, when compared with matched adjacent normal tissue. This loss of HP1ß expression may in part be caused by microRNA dysregulation. An example is miR-205, a microRNA that is abundantly upregulated in thyroid carcinomas and shown to reduce the expression of HP1ß. In contrast to HP1ß, HP1α expression was only reduced in metastatic carcinomas and poorly differentiated lesions. These results suggest the reduction of HP1ß followed by a decrease in HP1α contributes to the pathogenesis of thyroid carcinomas, and their loss is a potential marker of thyroid malignancy and metastatic potential, respectively.

Antiapoptotic protein Lifeguard is required for survival and maintenance of Purkinje and granular cells.

Lifeguard (LFG) is an inhibitor of Fas-mediated cell death and is highly expressed in the cerebellum. We investigated the biological role of LFG in the cerebellum in vivo, using mice with reduced LFG expression generated by shRNA lentiviral transgenesis (shLFG mice) as well as LFG null mice. We found that LFG plays a role in cerebellar development by affecting cerebellar size, internal granular layer (IGL) thickness, and Purkinje cell (PC) development. All these features are more severe in early developmental stages and show substantial recovery overtime, providing a remarkable example of cerebellar plasticity. In adult mice, LFG plays a role in PC maintenance shown by reduced cellular density and abnormal morphology with increased active caspase 8 and caspase 3 immunostaining in shLFG and knockout (KO) PCs. We studied the mechanism of action of LFG as an inhibitor of the Fas pathway and provided evidence of the neuroprotective role of LFG in cerebellar granule neurons (CGNs) and PCs in an organotypic cerebellar culture system. Biochemical analysis of the Fas pathway revealed that LFG inhibits Fas-mediated cell death by interfering with caspase 8 activation. This result is supported by the increased number of active caspase 8-positive PCs in adult mice lacking LFG. These data demonstrate that LFG is required for proper development and survival of granular and Purkinje cells and suggest LFG may play a role in cerebellar disorders.

PAX5 is expressed in small-cell lung cancer and positively regulates c-Met transcription.

PAX5 is a nuclear transcription factor required for B cell development, and its expression was evaluated in upper aerodigestive malignancies and pancreatic cancer by immunoblotting. The PAX5 protein expression was relatively strong in small-cell lung cancer (SCLC, 11/12); however, its expression was not detected in non-SCLC (NSCLC, n=13), mesothelioma (n=7), pancreatic (n=6), esophageal (n=6) and head and neck cancer cell lines (n=12). In comparison, PAX8 and PAX3 expressions were absent or non-detectable in SCLC cell lines; however, PAX8 was expressed in most of the tested NSCLC cell lines (13/13) and also frequently in all the other cell lines. We also detected frequent expressions of PAX2 and PAX9 protein in the various cell lines. Utilizing neuroendocrine tumor samples, we found that the frequency as well as the average intensity of the expression of PAX5 increased from pulmonary carcinoid (9%, moderate and strong PAX5 expression, n=44), to large-cell neuroendocrine carcinoma (LCNC, 27%, n=11) to SCLC (33%, n=76). FISH analysis revealed no translocations of the PAX5 gene, but polyploidy in some SCLC tumor tissues (6/37). We determined that PAX5 could regulate the transcription of c-Met using luciferase-coupled reporter and chromatin immunoprecipitation analysis. In addition, the phospho-c-Met (active form) and PAX5 were both localized to the same intra-nuclear compartment in hepatocyte growth factor treated SCLC cells and interacted with each other. Finally, we determined the therapeutic translational potential of PAX5 using PAX5 knockdown SCLC cells in conjunction with Topoisomerase 1 (SN38) and c-Met (SU11274) inhibitors. Loss of endogenous PAX5 significantly decreased the viability of SCLC cells, especially when combined with SN38 or SU11274, and maximum effect was seen when both inhibitors were used. Therefore, we propose that PAX5 could be an important regulator of c-Met transcription and a potential target for therapy in SCLC.

Identification of Novel Binding Partners for Transcription Factor Emx2.

The mammalian homolog of Drosophila empty spiracles 2 (Emx2) is a homeobox transcription factor that plays central roles in early development of the inner ear, pelvic and shoulder girdles, cerebral cortex, and urogenital organs. The role for Emx2 is best understood within the context of the development of the neocortical region of the cortex, where Emx2 is expressed in a high posterior-medial to low anterior-lateral gradient that regulates the partitioning of the neocortex into different functional fields that perform discrete computational tasks. Despite several lines of evidence demonstrating an Emx2 concentration-dependent mechanism for establishing functional areas within the developing neocortex, little is known about how Emx2 physically carries out this role. Although several binding partners for Emx2 have been identified (including Sp8, eIF4E, and Pbx1), no screens have been used to identify potential protein binding partners for this protein. We utilized a yeast two-hybrid screen using a library constructed from embryonic mouse cDNA in an attempt to identify novel binding partners for Emx2. This initial screen isolated two potential Emx2-binding partner proteins, Cnot6l and QkI-7. These novel Emx2-binding proteins are involved in multiple levels of mRNA metabolism that including splicing, mRNA export, translation, and destruction, thus making them interesting targets for further study.

Molecular detection of PPAR gamma rearrangements and thyroid carcinoma in preoperative fine-needle aspiration biopsies.

The pathologic diagnosis of thyroid follicular tumors is difficult, particularly in preoperative fine-needle aspiration biopsies. To investigate whether the molecular diagnosis of PPAR gamma rearrangements can detect thyroid carcinomas in fine-needle aspiration biopsies, we performed interphase fluorescence in situ hybridization on 24 thyroid fine-needle aspiration and 17 follow-up thyroidectomy specimens. Two of the 24 fine-needle aspiration biopsies contained PPAR gamma rearrangements, and both were diagnosed suggestive of a thyroid follicular neoplasm by cytology. The two corresponding thyroidectomies each contained PPAR gamma rearrangements in all tumor cells and, both were diagnosed follicular-patterned thyroid carcinomas-one a follicular carcinoma and the other a follicular variant of papillary carcinoma, the latter by majority of expert endocrine pathologists. Our experiments demonstrate that PPAR gamma rearrangements can detect a subset of follicular-patterned thyroid carcinomas in preoperative thyroid fine-needle aspiration biopsies. The ultimate utility of mutations such as PPAR gamma rearrangements in diagnosis of thyroid carcinoma must be proven by direct correlation of mutation status with thyroid tumor biology and not just with thyroid tumor morphology, a subjective and imprecise marker of clinical behavior. The application of specific mutations to preoperative diagnosis of thyroid carcinoma is predicted to improve the accuracy and reduce the costs of treating patients with thyroid tumors.

The PAX8/PPARgamma fusion oncoprotein transforms immortalized human thyrocytes through a mechanism probably involving wild-type PPARgamma inhibition.

Follicular thyroid carcinoma (FTC) frequently harbors the PAX8/PPARgamma fusion gene (PPFP); however, its oncogenic role and mechanism(s) of action remain undefined. We investigated PPFP's effects on cell growth, apoptosis, cell-cell, and cell-matrix interactions in immortalized human thyroid cells (Nthy-ori 3-1) and NIH 3T3 cells. PPFP expression increased the growth of transient and stable Nthy-ori transfectants ( approximately threefold by 72 h). There was an 8.4% increase of cells in the S+G2/M phase, a 7.8% decrease in cells in the G0+G1 phase and a 66% decline in apoptosis at 72 h. Stable Nthy-ori PPFP transfectants grew in soft agar, and PPFP-transfected NIH 3T3 cells exhibited efficient focus formation, suggesting loss of anchorage-dependent growth and contact inhibition, respectively. Overexpression of PPARgamma in Nthy-ori cells did not recapitulate PPFP's growth effects. Treatment of Nthy-ori cells with an irreversible PPARgamma inhibitor mimicked the growth-promoting effects of PPFP and co-expression of PPFP and PPARgamma blocked PPARgamma transactivation activity. Our data provide functional evidence that PPFP acts as an oncoprotein, whose transforming properties depend in part on inhibition of PPARgamma. Our data suggest that PPFP contributes to malignant transformation during FTC oncogenesis by acting on several cellular pathways, at least some of which are normally regulated by PPARgamma.

Inhibitory effects of peroxisome poliferator-activated receptor gamma on thyroid carcinoma cell growth.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor involved in such cellular processes as adipogenesis, inflammation, atherosclerosis, cell cycle control, apoptosis, and carcinogenesis. PPAR gamma gene mutations have been found in 4 of 55 sporadic colon cancers, and a chimeric PAX8-PPAR gamma 1 gene frequently generates a chromosomal translocation in thyroid follicular carcinomas, implicating PPAR gamma in tumor suppression. We investigated whether PPAR gamma is involved in the growth regulation of normal and tumor thyroid cells. We found no mutations in PPAR gamma exons 3 and 5 in human thyroid carcinoma cell lines and tissues. Moreover, 1 cell line (NPA) of 6 analyzed did not express PPAR gamma. Treatment of NPA with PPAR gamma agonists did not induce any inhibitory effect. Conversely, PPAR gamma agonists and PPAR gamma overexpression led to a drastic reduction of the cell growth rate in PPAR gamma-expressing thyroid carcinoma cells. Restoration of PPAR gamma expression in NPA cells induced cell growth inhibition; PPAR gamma agonists induced further inhibition. Growth inhibition induced by PPAR gamma agonists or by PPAR gamma gene overexpression in thyroid carcinoma cells was associated with increased p27 protein levels and apoptotic cell death. Should these data be confirmed, PPAR gamma could be a novel target for innovative therapy of thyroid carcinoma, particularly anaplastic carcinomas, which represent one of the most aggressive tumors in mankind and are unresponsive to conventional therapy.

RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma.

A series of 88 conventional follicular and Hürthle cell thyroid tumors were analyzed for RAS mutations and PAX8-PPAR gamma rearrangements using molecular methods and for galectin-3 and HBME-1 expression by immunohistochemistry. A novel LightCycler technology-based method was developed to detect point mutations in codons 12/13 and 61 of the H-RAS, K-RAS, and N-RAS genes. Forty-nine percent of conventional follicular carcinomas had RAS mutations, 36% had PAX8-PPAR gamma rearrangement, and only one (3%) had both. In follicular adenomas, 48% had RAS mutations, 4% had PAX8-PPAR gamma rearrangement, and 48% had neither. Follicular carcinomas with PAX8-PPAR gamma typically showed immunoreactivity for galectin-3 but not for HBME-1, tended to present at a younger patient age and be smaller size, and were almost always overtly invasive. In contrast, follicular carcinomas with RAS mutations most often displayed an HBME-1-positive/galectin-3-negative immunophenotype and were either minimally or overtly invasive. Hürthle cell tumors infrequently had PAX8-PPAR gamma rearrangement or RAS mutations. These results suggest that conventional follicular thyroid carcinomas develop through at least two distinct and virtually nonoverlapping molecular pathways initiated by either RAS point mutation or PAX8-PPAR gamma rearrangement.

PAX8-PPARgamma rearrangement in thyroid tumors: RT-PCR and immunohistochemical analyses.

A PAX8-PPARgamma rearrangement has been recently identified in follicular thyroid carcinomas, but not in follicular adenomas or other thyroid tumors. We report here the analyses of PAX8-PPARgamma in a series of 118 thyroid tumors using a newly developed RT-PCR assay to detect this rearrangement in frozen and paraffin-embedded tissues and using immunostaining with a PPARgamma antibody. PAX8-PPARgamma was detected by RT-PCR in eight of 15 (53%) follicular carcinomas and two of 25 (8%) follicular adenomas but not in 35 papillary carcinomas (including 12 follicular variants), 12 Hurthle cell carcinomas, 12 Hurthle cell adenomas, two anaplastic carcinomas, one poorly differentiated carcinoma, or 16 hyperplastic nodules. The prevalence was higher in follicular carcinomas from patients with a history of radiation exposure (three of three). Strong, diffuse nuclear immunostaining with the PPARgamma antibody correlated with the presence of PAX8-PPARgamma detected by RT-PCR. Most sporadic follicular carcinomas positive for PAX8-PPARgamma were overtly invasive, whereas tumors lacking the rearrangement were predominantly minimally invasive. The two follicular adenomas positive for PAX8-PPARgamma had trabecular growth pattern and thick capsule, but no invasion, and thus may represent "pre-invasive" follicular carcinomas. The absence of PAX8-PPARgamma rearrangements in Hurthle cell tumors and papillary thyroid carcinomas highlights the differences in the molecular pathogenesis of these thyroid tumors.

Molecular events in follicular thyroid tumors.

Knowledge of the molecular events that govern human thyroid tumorigenesis has grown considerably in the past ten years. Key genetic alterations and new oncogenic pathways have been identified. Molecular genetic aberrations in thyroid carcinomas bear noteworthy resemblance to those in acute myelogenous leukemias. Thyroid carcinomas and myeloid leukemias both possess transcription factor gene rearrangements-PPARgamma-related translocations in thyroid carcinoma and RARalpha-related and CBF-related translocations (amongst others) in myeloid leukemia. PPARgamma and RARalpha are closely related members ofthe same nuclear receptor subfamily, and the PML-RARalpha and PAX8-PPARgamma fusion proteins both function as dominant negative inhibitors of their wild-type parent proteins. Thyroid carcinomas and myeloid leukemias also both harbor NRAS mutations (15-25% of both cancers) and receptor tyrosine kinase mutations--RET mutations in thyroid carcinomas and FLT3 mutations in myeloid leukemias. The NRAS and tyrosine receptor kinase mutations are not observed in the same thyroid carcinoma or leukemia patients, suggesting that multiple initiating pathways exist in both. Lastly, thyroid carcinomas and myeloid leukemias possess p53 mutations at relatively low frequency (10-15%) in patients who tend to be older and have more aggressive, therapy resistant disease. Such parallels are unlikely to occur by chance alone and argue that common mechanisms underlie these diverse epithelial and hematologic cancers. The comparison of thyroid carcinomas and myeloid leukemias may highlight areas of thyroid cancer investigation worthy of further focus. For example, few collaborating mutations have been defined in thyroid carcinomas even though they play a clear role in myeloid leukemias, as exemplified by RARalpha rearrangements and FLT3 mutations that together dictate the promyleocytic leukemia phenotype. Functional interactions between collaborating mutations are possible at multiple levels, and it is tempting to speculate that some thyroid carcinomas might develop through an unique combination or co-activation of RET and RAS and/or RET and PPARgamma (and/or other) signaling systems. In fact, the ELE1-RET (PTC3) fusion protein contains the ELE1 nuclear receptor co-activator domain and it appears to physically associate with and inhibit wild-type PPARgamma in some papillary carcinomas. The similarities of the fusion proteins in thyroid carcinoma and myeloid leukemia suggest that a more directed search for fusion genes in non-thyroid carcinomas is warranted. In fact, novel fusion genes have been identified recently in aggressive midline, secretory breast, and renal cell carcinomas, although the epithelial nature of the latter is not well-documented. Interestingly, these cancers all tend to present more frequently in adolescence and young adulthood in a manner similar to thyroid and myeloid malignancies that have fusion genes. The analyses of cancers that present earlier in life may enhance fusion gene recognition in other carcinoma types. Definition and biologic characterization of the precursor cells that give rise to thyroid carcinoma will also be important. Myeloid leukemias are thought to arise from stem/progenitor cells that acquire disturbed self-renewal and differentiation capacities but retain characteristics of the myeloid lineages. Although the presence of comparable stem/progenitor cells in the thyroid are not defined, distinct thyroid cancer lineages and patterns of differentiation exist and candidate stem/progenitor cells such as the p63-immunoreactive solid cell nests are apparent. A last important area is development of molecular-based therapies for thyroid carcinoma patients resistant to standard radio-iodine treatment. Treatments for such cancers are limited and pathways defined by thyroid cancer mutations are prime targets for pharmacologic interventions with molecular inhibitors. Tyrosine kinase inhibitors and nuclear receptor ligands have proven dramatically effective in some myeloid leukemia patients. Various molecular inhibitors are being investigated now in thyroid cancer models. Such developments predict that the thyroid cancer model will continue to provide biologic insights into human carcinoma biology and that improved pathologic diagnosis and treatment for thyroid cancer patients sit on the not too distant horizon.

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA.

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2xRBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.

Lhx2 specifies regional fate in Emx1 lineage of telencephalic progenitors generating cerebral cortex.

Cerebral cortex is comprised of regions, including six-layer neocortex and three-layer olfactory cortex, generated by telencephalic progenitors of an Emx1 lineage. The mechanism specifying region-specific subpopulations in this lineage is unknown. We found that the LIM homeodomain transcription factor Lhx2 in mice, expressed in graded levels by progenitors, determines their regional identity and fate decisions to generate neocortex or olfactory cortex. Deletion of Lhx2 with Emx1-cre at embryonic day 10.5 (E10.5) altered the fates of progenitors, causing them to generate three-layer cortex, phenocopying olfactory cortex rather than lateral neocortex. Progenitors did not generate ectopic olfactory cortex following Lhx2 deletion at E11.5. Thus, Lhx2 regulates a regional-fate decision by telencephalic progenitors during a critical period that ends as they differentiate from neuroepithelial cells to neuronogenic radial glia. These findings establish a genetic mechanism for determining regional-fate in the Emx1 lineage of telencephalic progenitors that generate cerebral cortex.

Peroxisome proliferator-activated receptor-delta induces cell proliferation by a cyclin E1-dependent mechanism and is up-regulated in thyroid tumors.

Peroxisome proliferator-activated receptors (PPARs) are lipid-sensing nuclear receptors that have been implicated in multiple physiologic processes including cancer. Here, we determine that PPARdelta induces cell proliferation through a novel cyclin E1-dependent mechanism and is up-regulated in many human thyroid tumors. The expression of PPARdelta was induced coordinately with proliferation in primary human thyroid cells by the activation of serum, thyroid-stimulating hormone/cyclic AMP, or epidermal growth factor/mitogen-activated protein kinase mitogenic signaling pathways. Engineered overexpression of PPARdelta increased thyroid cell number, the incorporation of bromodeoxyuridine, and the phosphorylation of retinoblastoma protein by 40% to 45% in just 2 days, one usual cell population doubling. The synthetic PPARdelta agonist GW501516 augmented these PPARdelta proliferation effects in a dose-dependent manner. Overexpression of PPARdelta increased cyclin E1 protein by 9-fold, whereas knockdown of PPARdelta by small inhibitory RNA reduced both cyclin E1 protein and cell proliferation by 2-fold. Induction of proliferation by PPARdelta was abrogated by knockdown of cyclin E1 by small inhibitory RNA in primary thyroid cells and by knockout of cyclin E1 in mouse embryo fibroblasts, confirming a cyclin E1 dependence for this PPARdelta pathway. In addition, the mean expression of native PPARdelta was increased by 2-fold to 5-fold (P < 0.0001) and correlated with that of the in situ proliferation marker Ki67 (R = 0.8571; P = 0.02381) in six different classes of benign and malignant human thyroid tumors. Our experiments identify a PPARdelta mechanism that induces cell proliferation through cyclin E1 and is regulated by growth factor and lipid signals. The data argue for systematic investigation of PPARdelta antagonists as antineoplastic agents and implicate altered PPARdelta-cyclin E1 signaling in thyroid and other carcinomas.

CREB3L2-PPARgamma fusion mutation identifies a thyroid signaling pathway regulated by intramembrane proteolysis.

The discovery of gene fusion mutations, particularly in leukemia, has consistently identified new cancer pathways and led to molecular diagnostic assays and molecular-targeted chemotherapies for cancer patients. Here, we report our discovery of a novel CREB3L2-PPARgamma fusion mutation in thyroid carcinoma with t(3;7)(p25;q34), showing that a family of somatic PPARgamma fusion mutations exist in thyroid cancer. The CREB3L2-PPARgamma fusion encodes a CREB3L2-PPARgamma fusion protein that is composed of the transactivation domain of CREB3L2 and all functional domains of PPARgamma1. CREB3L2-PPARgamma was detected in <3% of thyroid follicular carcinomas. Engineered overexpression of CREB3L2-PPARgamma induced proliferation by 40% to 45% in primary human thyroid cells, consistent with a dominant oncogenic mechanism. Wild-type CREB3L2 was expressed in the thyroid as a bZIP transcription factor with a transmembrane domain that has flanking S1P and S2P proteolytic cleavage sites. Native CREB3L2 was cleaved to nuclear CREB3L2 by regulated intramembrane proteolysis in normal thyroid cells that expressed the S1P and S2P proteases. Nuclear CREB3L2 stimulated transcription 8-fold from the EVX1 cyclic AMP (cAMP) response element in the absence of cAMP, whereas CREB3L2-PPARgamma inhibited transcription 6-fold from EVX1 in the same experiments. CREB3L2-PPARgamma also inhibited 4-fold the expression of thyroglobulin, a native cAMP-responsive gene, in primary thyroid cells treated with thyroid-stimulating hormone. Our findings identify a novel CREB3L2-PPARgamma gene fusion mutation in thyroid carcinoma and reveal a thyroid signaling pathway that is regulated by intramembrane proteolysis and disrupted in cancer.

Cortical area size dictates performance at modality-specific behaviors.

The mammalian neocortex is organized into unique areas that serve functions such as sensory perception and modality-specific behaviors. The sizes of primary cortical areas vary across species, and also within a species, raising the question of whether area size dictates behavioral performance. We show that adult mice genetically engineered to overexpress the transcription factor EMX2 in embryonic cortical progenitor cells, resulting in reductions in sizes of somatosensory and motor areas, exhibit significant deficiencies at tactile and motor behaviors. Even increasing the size of sensorimotor areas by decreasing cortical EMX2 levels can lead to diminished sensorimotor behaviors. Genetic crosses that retain ectopic Emx2 transgene expression subcortically but restore cortical Emx2 expression to wild-type levels also restore cortical areas to wild-type sizes and in parallel restore tactile and motor behaviors to wild-type performance. These findings show that area size can dictate performance at modality-specific behaviors and suggest that areas have an optimal size, influenced by parameters of its neural system, for maximum behavioral performance. This study underscores the importance of establishing during embryonic development appropriate levels of regulatory proteins that determine area sizes, thereby influencing behavior later in life.

Ventralized dorsal telencephalic progenitors in Pax6 mutant mice generate GABA interneurons of a lateral ganglionic eminence fate.

The transcription factor Pax6 is expressed by progenitors in the ventricular zone (VZ) of dorsal telencephalon (dTel), which generate all cortical glutamatergic neurons, but not by progenitors in the medial ganglionic eminence (MGE), which generate cortical GABAergic interneurons (GABA INs), or the lateral ganglionic eminence (LGE), which generate GABA INs that normally migrate to the olfactory bulb. We show that perinatally, Pax6(sey/sey) mice, which lack functional Pax6 protein, have large subpial ectopias in dTel and ventral telencephalon connected by cell streams arising from an aberrant paraventricular ectopia found throughout dTel. The subpial and paraventricular ectopias and connecting cell streams are comprised of postmitotic neurons expressing markers for GABA INs characteristic of a LGE fate. Marker analyses show that dTel VZ progenitors in Pax6 mutants are progressively ventralized, acquiring expression of regulatory genes normally limited to GE progenitors; by midneurogenesis, the entire dTel VZ exhibits ventralization. This ventralization of the dTel VZ is paralleled by the expression of markers for GABA INs superficial to it, suggesting that it ectopically produces GABA INs, leading to their ectopias and a thinner cortical plate due to diminished production of glutamatergic neurons. Genetic lineage tracing demonstrates that the GABA INs comprising the ectopias are from a cortical Emx1 lineage generated in the dTel VZ, definitively showing that dTel progenitors and progeny acquire a ventral, GE, fate in Pax6 mutants. Thus, Pax6 delimits the appropriate proliferative zone for GABA INs and regulates their numbers and distributions by repressing the ventral fates of dTel progenitors and progeny.

PIKE-A is amplified in human cancers and prevents apoptosis by up-regulating Akt.

PIKE-A (PIKE-activating Akt), an isoform of PIKE GTPase that enhances phosphatidylinositol 3-kinase (PI3-kinase) activity, specifically binds to active Akt but not PI3-kinase. PIKE-A stimulates Akt activity in a GTP-dependent manner and promotes invasiveness of cancer cell lines. Here, we show that PIKE-A is amplified in a variety of human cancers and that amplified PIKE-A directly stimulates Akt and inhibits apoptosis compared to cells with normal PIKE-A copy number. Overexpression of PIKE-A wild-type but not dominant-negative mutant stimulates Akt activity and prevents apoptosis. Moreover, knockdown of PIKE-A diminishes Akt activity and increases apoptosis. Our findings suggest that PIKE-A amplification contributes to cancer cell survival and progression by inhibiting apoptosis through up-regulating Akt.

A homolog of FBP2/KSRP binds to localized mRNAs in Xenopus oocytes.

A Xenopus oocyte expression library was screened for proteins that bind to the 340-nucleotide localization element of Vg1 mRNA. Four different isolates encoded a Xenopus homolog of the human transcription factor, FUSE-binding protein 2 (FBP2). This protein has been independently identified as the splicing regulatory factor KSRP. The only significant difference between the Xenopus protein, designated VgRBP71, and KSRP is the absence of a 58 amino acid segment near the N-terminal of the former. In vivo binding assays show that VgRBP71 is associated with mRNAs localized to either the vegetal or animal hemispheres, but was not found with control mRNAs. Unlike other factors that bind to the localization element of Vg1 mRNA, VgRBP71 does not accumulate at the vegetal cortex with the mRNA; rather, it is present in the nucleus and throughout the cytoplasm at all stages of oogenesis. Cytoplasmic VgRBP71 appears to be most concentrated at the cell cortex. VgRBP71 interacts with Prrp, another protein that binds to the Vg1 localization element; this association does not require the presence of Vg1 mRNA.

PIKE (phosphatidylinositol 3-kinase enhancer)-A GTPase stimulates Akt activity and mediates cellular invasion.

Akt/PKB is a crucial regulator of diverse cellular processes and contributes to cancer progression. Activation of Akt is essentially dependent on phosphatidylinositol (PI) 3-kinase signaling. Here, we describe a novel mediator of Akt that is independent of PI 3-kinase. This mediator, PIKE-A, is a PIKE isoform and contains GTPase, pleckstrin homology, ArfGAP, and ankyrin repeats domains. PIKE-A directly binds to activated Akt but not PI 3-kinase in a guanine nucleotide-dependent way and stimulates the kinase activity of Akt. Overexpression of PIKE-A enhances Akt activity and promotes cancer cell invasion, whereas dominant-negative PIKE-A and PIKE-A knockdown markedly inhibit these processes. Our results demonstrate that PIKE-A is a physiologic regulator of Akt and an oncogenic effector of cell invasion.

Genetic and biological subgroups of low-stage follicular thyroid cancer.

Investigations of cancer-specific gene rearrangements have increased our understanding of human neoplasia and led to the use of the rearrangements in pathological diagnosis of blood cell and connective tissue malignancies. Here, we have investigated 3p25 rearrangements of the peroxisome proliferator-activated receptor gamma (PPAR gamma) gene in follicular epithelial tumors of the human thyroid gland. Eleven of 42 (26%) low-stage follicular carcinomas, 0 of 40 follicular adenomas, 1 of 30 Hurthle cell carcinomas, 1 of 90 papillary carcinomas, and 0 of 10 nodular goiters had 3p25 rearrangements by interphase fluorescence in situ hybridization. All 11 follicular carcinomas with 3p25 rearrangement exhibited strong, diffuse nuclear immunoreactivity for PPAR gamma, consistent with expression of PPAR gamma fusion protein. Twelve of 42 (29%) low-stage follicular carcinomas had 3p25 aneusomy without PPAR gamma rearrangement (P = 0.01), suggesting that PPAR gamma rearrangement and aneuploidy are independent early events in follicular cancer. Eleven of 12 follicular carcinomas with 3p25 aneusomy exhibited no PPAR gamma immunoreactivity, supporting the existence of two independent pathways. Follicular carcinoma patients with PPAR gamma rearrangement more frequently had vascular invasion (P = 0.01), areas of solid/nested tumor histology (P < 0.001), and previous non-thyroid cancers (P < 0.01) compared with follicular carcinoma patients without PPAR gamma rearrangement. Our experiments identify genetic subgroups of low-stage follicular thyroid cancer and provide evidence that follicular carcinomas with PPAR gamma rearrangement are a distinct biological entity. The findings support a model in which separate genetic alterations initiate distinct pathways of oncogenesis in thyroid carcinoma subtypes.