Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously 'undruggable' oncogenic or hyperactive Ras

LOSS OF JAK2 REGULATION VIA VHL-SOCS1 E3 UBIQUITIN HETEROCOMPLEX UNDERLIES CHUVASH POLYCYTHEMIA

Chuvash polycythemia (CP) is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the von Hippel-Lindau (VHL) gene whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark features of CP such as hypersensitivity to erythropoietin are unclear. Here, we show that VHL directly binds suppressor of cytokine signalling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated (p)JAK2 for ubiquitin-mediated destruction. In contrast, CP-associated VHL mutants have altered affinity for SOCS1 and fail to engage and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reverses the disease phenotype in vhlR200W/R200W knock-in mice, a model that faithfully recapitulates human CP. These results reveal VHL as a SOCS1-cooperative negative regulator of JAK2 and provide compelling biochemical and preclinical evidence for JAK2- targeted therapy in CP patients

Regulation of Cellular Oxygen Sensing Pathways by VHL

Erythropoiesis represents a vital physiologic process that can be adjusted to combat compromised oxygen availability, otherwise known as hypoxia. The canonical response to adapt to hypoxia is mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). When oxygen is available HIFalpha subunits are hydroxylated by prolyl hydroxylase enzymes (PHD1-3) to allow for binding to the von Hippel-Lindau (VHL) tumour suppressor protein, which serves as the substrate recognition subunit of an E3-ubiquitin ligase complex that ultimately targets proteins for proteasome-mediated degradation. The work presented in this thesis demonstrates a novel role of VHL in recruiting DCNL1 to initiate E3-ubiquitin ligase enzymatic activity which, in essence, allows for the degradation of HIFalpha to commence. In hypoxia, hydroxylation does not take place and as a result HIFalpha can promote the transactivation of a repertoire of genes that aid in adaptation to hypoxia including one which encodes the glycoprotein hormone erythropoietin (EPO). EPO binds to EPO receptor (EPOR) expressed on erythroid progenitor cells to promote their proliferation and differentiation into red blood cells. An erythroid specific oxygen sensing pathway is elucidated in this thesis wherein EPOR is hydroxylated by PHD3 to promote VHL-mediated degradation in a manner analogous to HIFalpha. Combined, these findings build upon our fundamental understanding of how cells detect and counter hypoxic stress.Ph.D.2017-03-31 00:00:0

SOCS-1 Mediates Ubiquitylation and Degradation of GM-CSF Receptor

<div><p>Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the related cytokines interleukin (IL)-3 and IL-5 regulate the production and functional activation of hematopoietic cells. GM-CSF acts on monocytes/macrophages and granulocytes, and several chronic inflammatory diseases and a number of haematological malignancies such as Juvenile myelomonocytic leukaemia (JMML) are associated with deregulated GM-CSF receptor (GMR) signaling. The downregulation of GMR downstream signaling is mediated in part by the clearance of activated GMR via the proteasome, which is dependent on the ubiquitylation of βc signaling subunit of GMR via an unknown E3 ubiquitin ligase. Here, we show that suppressor of cytokine signaling 1 (SOCS-1), best known for its ability to promote ubiquitin-mediated degradation of the non-receptor tyrosine kinase Janus kinase 2 (JAK2), also targets GMRβc for ubiquitin-mediated degradation and attenuates GM-CSF-induced downstream signaling.</p></div

SOCS-1 preferentially binds to and ubiquitylates GMRβc.

<p>(A) HEK293 cells transfected with plasmids encoding GMRα and βc in combination with Flag-SOCS-1, -2, -3 or an empty plasmid (mock) were lysed, immunoprecipitated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. (B) HEK293 cells transfected with plasmids encoding GMRα, βc and HA-ubiquitin (HA-Ub) in combination with Flag-SOCS-1, -3 or an empty plasmid (mock) were treated for 4 h with (+) MG132 or (−) DMSO then lysed, immunoprecipiated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. (C) HEK293 cells transfected with plasmids encoding GMRα and βc in combination with Flag-SOCS-1, -SOCS-1▵SOCS-Box mutant, or empty plasmid (mock) were treated for 4 h with (+) MG132 or (–) DMSO then lysed, immunoprecipiated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. WCE: whole cell extract.</p

Knockdown of endogenous SOCS-1 in TF-1 cells promotes GMRβc stabilization and GM-CSF-induced downstream signaling.

<p>(A) Serum and cytokine starved TF-1 cells were treated with GM-CSF for the indicated times, lysed, immunoprecipitated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. (B) TF-1 cells transduced with lentivirus-shSOCS-1 or non-targeting scrambled shRNA (shScr) were lysed and immunoblotted with the indicated antibodies. (C) TF-1-shSOCS-1 and TF-1-shScr cells were treated with (+) MG132 or (–) DMSO for 4 h prior to immunoprecipitation (IP) with anti-GMRβc antibody and subsequent immunoblot analysis using the indicated antibodies. (D) Serum and cytokine starved TF-1-ShScr or TF-1-ShSOCS-1 cells were treated with GM-CSF for the indicated times, lysed and immunoblotted with the indicated antibodies. WCE: whole cell extract.</p

Deregulation of E2-EPF Ubiquitin Carrier Protein in Papillary Renal Cell Carcinoma

Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC