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

Increased mRNA expression of CDKN2A is a transcriptomic marker of clinically aggressive meningiomas

Homozygous deletion of CDKN2A/B was recently incorporated into the World Health Organization classification for grade 3 meningiomas. While this marker is overall rare in meningiomas, its relationship to other CDKN2A alterations on a transcriptomic, epigenomic, and copy number level has not yet been determined. We therefore utilized multidimensional molecular data of 1577 meningioma samples from 6 independent cohorts enriched for clinically aggressive meningiomas to comprehensively interrogate the spectrum of CDKN2A alterations through DNA methylation, copy number variation, transcriptomics, and proteomics using an integrated molecular approach. Homozygous CDKN2A/B deletions were identified in only 7.1% of cases but were associated with significantly poorer outcomes compared to tumors without these deletions. Heterozygous CDKN2A/B deletions were identified in 2.6% of cases and had similarly poor outcomes as those with homozygous deletions. Among tumors with intact CDKN2A/B (without a homozygous or heterozygous deletion), we found a distinct difference in outcome based on mRNA expression of CDKN2A, with meningiomas that had elevated mRNA expression (CDKN2Ahigh) having a significantly shorter time to recurrence. The expression of CDKN2A was independently prognostic after accounting for copy number loss and consistently increased with WHO grade and more aggressive molecular and methylation groups irrespective of cohort. Despite the discordant and mutually exclusive status of the CDKN2A gene in these groups, both CDKN2Ahigh meningiomas and meningiomas with CDKN2A deletions were enriched for similar cell cycle pathways but at different checkpoints. High mRNA expression of CDKN2A was also associated with gene hypermethylation, Rb-deficiency, and lack of response to CDK inhibition. p16 immunohistochemistry could not reliably differentiate between meningiomas with and without CDKN2A deletions but appeared to correlate better with mRNA expression. These findings support the role of CDKN2A mRNA expression as a biomarker of clinically aggressive meningiomas with potential therapeutic implications

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

Aldosterone and its Antagonists Modulate Elastin Deposition in the Heart

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.Ph

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

Liquid Biomarkers for Improved Diagnosis and Classification of CNS Tumors

Liquid biopsy, as a non-invasive technique for cancer diagnosis, has emerged as a major step forward in conquering tumors. Current practice in diagnosis of central nervous system (CNS) tumors involves invasive acquisition of tumor biopsy upon detection of tumor on neuroimaging. Liquid biopsy enables non-invasive, rapid, precise and, in particular, real-time cancer detection, prognosis and treatment monitoring, especially for CNS tumors. This approach can also uncover the heterogeneity of these tumors and will likely replace tissue biopsy in the future. Key components of liquid biopsy mainly include circulating tumor cells (CTC), circulating tumor nucleic acids (ctDNA, miRNA) and exosomes and samples can be obtained from the cerebrospinal fluid, plasma and serum of patients with CNS malignancies. This review covers current progress in application of liquid biopsies for diagnosis and monitoring of CNS malignancies

Liquid Biomarkers for Improved Diagnosis and Classification of CNS Tumors

Liquid biopsy, as a non-invasive technique for cancer diagnosis, has emerged as a major step forward in conquering tumors. Current practice in diagnosis of central nervous system (CNS) tumors involves invasive acquisition of tumor biopsy upon detection of tumor on neuroimaging. Liquid biopsy enables non-invasive, rapid, precise and, in particular, real-time cancer detection, prognosis and treatment monitoring, especially for CNS tumors. This approach can also uncover the heterogeneity of these tumors and will likely replace tissue biopsy in the future. Key components of liquid biopsy mainly include circulating tumor cells (CTC), circulating tumor nucleic acids (ctDNA, miRNA) and exosomes and samples can be obtained from the cerebrospinal fluid, plasma and serum of patients with CNS malignancies. This review covers current progress in application of liquid biopsies for diagnosis and monitoring of CNS malignancies