YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Evasion of p53 and G2/M checkpoints are characteristic of Hh-driven basal cell carcinoma.

Basal cell carcinoma (BCC), the most common type of cancer, is characterized by aberrant Hedgehog (Hh) pathway activity. Mutations in pathway components, such as PATCHED1 (PTCH1), are commonly found in BCC. While the tumor suppressor role of PTCH1 in BCC is well established, how Hh pathway activation disrupts normal skin homeostasis to promote BCC formationremains poorly understood. Like Ptc1, Sufu is a major negative regulator of the Hh pathway. Previously, we showed that inactivation of Sufu in the skin does not result in BCC formation. Why loss of Ptc1, but not Sufu, in the epidermis induces BCC formation is unclear. In this report, we utilized gene expression profiling to identify biological pathways and processes that distinguish Sufu from Ptc1 mutants, and discovered a novel role for Sufu in cell cycle regulation. We demonstrated that the Hh pathway activation inSufu and Ptc1 mutant skin is associated with abnormal cell cycle entry, ectopic expression of D-type cyclins and increasedDNA damage. However, despite the presence of DNA damage, p53 stabilization was impaired in the mutant skin. Alternative mechanism to halt genomic instability is the activation of G2/M cell cycle checkpoint, which can occur independent of p53. We found that while Ptc1 mutant cells continue to cycle, which would favor genomic instability, loss of Sufu results in G2/M cell cycle arrest.This finding may explain why inactivation of Sufu is not sufficient to drive BCC formation. Taken together, these studies revealed a unique role for Sufu in G2/M phase progression, and uncovered the molecular and cellular features associated with Hh-driven BCC.

A protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression.

By analyzing public data sets of gene expression in human breast cancers we observed that increased levels of transcripts encoding the planar cell polarity (PCP) proteins SCRIB and VANGL1 correlate with increased risk of patient relapse. Experimentally, we found that reducing expression of SCRIB by short-hairpin RNAs (shRNAs) reduces the growth of human breast cancer cells in xenograft assays. To investigate SCRIB-associated proteins that might participate in the responses of breast cancer cells to altered levels of SCRIB, we used mass spectrometry and confocal microscopy. These studies reveal that SCRIB is present in at least two unique protein complexes: (1) a complex of SCRIB, ARHGEF, GIT and PAK (p21-activated kinase), and (2) a complex of SCRIB, NOS1AP and VANGL. Focusing on NOS1AP, we observed that NOS1AP colocalizes with both SCRIB and VANGL1 along cellular protrusions in metastatic breast cancer cells, but does not colocalize with either SCRIB or VANGL1 at cell junctions in normal breast cells. We investigated the effects of shRNA-mediated knockdown of NOS1AP and SCRIB in vitro, and found that reducing NOS1AP and SCRIB slows breast cancer cell migration and prevents the establishment of leading-trailing polarity. We also find that reduction of NOS1AP enhances anchorage-independent growth. Collectively these data point to the relevance of NOS1AP and SCRIB protein complexes in breast cancer.

Biochemical and biophysical demonstration of GPCR oligomerization in mammalian cells.

In contrast to other families of cell surface receptors, like tyrosine kinase receptors, for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or hetero- oligomers could have important implications for the development and screening of new drugs. The major evidences supporting the idea of GPCR oligomerization come from indirect biochemical or pharmacological experiments. Here we report, using traditional co-immunoprecipitation methods, the existence of differentially epitope-tagged beta2-adrenergic receptor (beta2AR) oligomers in mammalian HEK-293 cells. Moreover, we validate the existence of receptor oligomers in living cells by a new Bioluminescence Resonance Energy Transfer (BRET) technique. Our results clearly demonstrate the presence of constitutive beta2AR oligomers in living cells that can be modulated by the selective adrenergic agonist isoproterenol, suggesting a pertinent physiological role for GPCR oligomerization.

Functional significance of oligomerization of G-protein-coupled receptors.

In contrast to other families of cell surface receptors for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or heterodimers could have important implications for the development and screening of new drugs.

Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants.

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.

Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.

Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis.

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.