Angiogenesis: PI3K subunits branch out

The catalytic subunits of class IA phosphoinositide 3-kinases (PI3K) are activated by distinct signals and have discrete roles in angiogenesis and vascular remodeling.

Class IA phosphoinositide 3-kinases (PI3Ks) are dimeric enzymes composed of a catalytic p110α, p110β or p110δ subunit bound to a regulatory p85 subunit. PI3Ks regulate a myriad of cellular functions, including angiogenesis, but the relative contribution of the different catalytic subunits towards this process has not yet been extensively characterized. In Nature, Graupera et al. analyze the activity of the catalytic p110 subunits in endothelial cells and find that p110α and p110β are activated by distinct signals and have discrete roles in angiogenesis.

The loss of p110α causes embryonic lethality due to defects in growth and angiogenesis. Replacement of endogenous p110α with a kinase dead variant (p110α^D933A/D933A), or targeted endothelial deletion of p110α also resulted in embryonic lethality and caused angiogenesis and vascular remodeling defects. Conversely, deletion of p110β in endothelial cells resulted in viable mice with no vascular defects, which is surprising given that p110β knockout animals die at the blastocyst stage of embryonic development. Inactivation of p110δ did not affect viability, likely because p110δ is predominantly expressed in leukocytes. Therefore, p110α plays a unique and important role in angiogenesis.

Aortic ring explants from p110α^D933A/WT heterozygous mice showed reduced vascular endothelial growth factor A (VEGF-A)-stimulated vessel outgrowth and Akt phosphorylation. Pharmacologic inhibition of p110α also inhibited outgrowth and tube formation in vitro, whereas genetic or pharmacologic inhibition of p110β or p110δ had no effect, suggesting that VEGF-A signals predominately through p110α to affect angiogenesis. In contrast, microvessel outgrowth and Akt phosphorylation stimulated by G-protein coupled receptor (GPCR) agonists was reduced in aortic rings from p110β-deficient, but not p110α^D933A/WT mice. These findings support the authors' previous observation that p110β predominately functions downstream of GPCRs.

VEGF-A signaling during angiogenesis promotes survival, proliferation and cell migration. Loss of p110α did not affect proliferation or survival, but did reduce the speed and total distance of migration in mouse and human endothelial cells in vitro, as well as retarding angiogenesis and migration in the mouse retina and embryonic hindbrain in vivo. Rho GTPases are key mediators of cell migration, and genetic or pharmacologic inactivation of p110α reduced RhoA activity and caused aberrant cell detachment — an activity regulated by RhoA. Exogenous expression of constitutively active RhoA in p110α^D933A/WT mouse cardiac endothelial cells rescued this defect.

These data show that p110α and p110β have highly specialized roles in angiogenesis. p110α transmits signals from VEGF-A to promote migration, whereas p110β functions downstream of GPCRs. The unique function of p110α in regulating endothelial cell motility supports the importance of this protein over p110β and p110δ in vascular remodeling and angiogenesis.

Emily J. Chenette
Signaling Gateway

Original Reference:
Graupera M. et al.
Angiogenesis selectively requires the p110α isoform of PI3K to control endothelial cell migration
Nature 453, 662-666 (2008)
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Colon cancer: Analyzing Ras specificity

Active N-Ras and K-Ras have different effects in colon cancer: K-Ras promotes proliferation and suppresses differentiation, whereas N-Ras protects cells from chemically induced apoptosis.

Colon cancer affects nearly 10% of the population worldwide. A significant proportion of colon adenocarcinomas express mutationally-activated Ras small GTPases. Despite their sequence and functional similarity, oncogenic K-Ras mutations are associated with malignant transformation, whereas constitutively active N-Ras is detected in the later stages of cancer. The basis for this differential activation has remained unclear. In Nature Genetics, Tyler Jacks and colleagues now examine the effect of K-Ras and N-Ras activation in colon cancer and report that these proteins promote different physiological responses.

Mice were engineered to express activated N-Ras (N-Ras^G12D) or K-Ras (K-Ras^G12D) from their respective endogenous promoters in the colonic epithelium. Active Mek was detected in cells that expressed K-Ras^G12D, but not N-Ras^G12D. Akt was downregulated in cells that expressed K-Ras or N-Ras, but there was no detectable alteration of other Ras effector pathways. Neither N-Ras^G12D nor K-Ras^G12D was sufficient to initiate tumor formation, although K-Ras^G12D expression caused Mek-dependent hyperplasia. Interestingly, N-Ras^G12D, but not K-Ras^G12D conferred resistance to chemically induced apoptosis, suggesting that N-Ras may function to suppress apoptosis in tumorigenesis.

Loss of the adenomatous polyposis coli (APC) tumor suppressor is thought to be an early event in colon cancer. Expression of K-Ras^G12D accelerated adenocarcinoma progression in APC-mutant mice, whereas N-Ras^G12D expression did not affect tumor development. In contrast to APC-deficient tumors, APC-deficient K-Ras^G12D-positive tumors expressed Mcm6 — a marker for undifferentiated cells — and the putative intestinal epithelial stem cell marker Musashi-1 (Msi1), suggesting that activated K-Ras suppresses differentiation. The poorly differentiated areas of K-Ras-positive tumors also expressed high levels of β-catenin.

Although pharmacologic inhibition of Mek reverted hyperplasia in colon epithelium, it did not affect tumorigenesis in the APC-deficient, K-Ras^G12D-positive mouse model or inhibit the growth of cultured human colon cancer cells. A library of small-molecule inhibitors of Ras effectors was screened to determine which K-Ras effectors promoted tumorigenesis. Pharmacological inhibition of Raf slowed cell growth, suggesting that Raf is a critical mediator of oncogenic K-Ras in vivo. The Raf isoform responsible for promoting hyperplasia was not identified; however, B-Raf mutations have been detected in a subset of human colon adenocarcinomas that do not also harbor K-Ras mutations.

In this mouse model of colon cancer, oncogenic K-Ras promotes proliferation and differentiation, whereas N-Ras protects cells from apoptosis. The finding that Mek inhibition in APC-deficient, K-Ras^G12D-positive tumors had no effect on tumor growth suggests that other signaling pathways downstream of Raf mediate tumorigenesis. As Mek is the canonical Raf target, additional studies are necessary to uncover Mek-independent signaling pathways responsible for proliferation.

Emily J. Chenette
Signaling Gateway

Original Reference:
Haigis, K. M. et al.
Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon
Nature Genetics 40, 600-608 (2008)
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Calcium signaling: Compounded aggregation

A screen for compounds that regulate autophagy has identified a cyclical inhibitory pathway in which cyclic AMP (cAMP) signaling increases intracellular calcium levels and stimulates calpain-mediated activation of Gα_s, which raises cAMP levels.

Protein aggregate formation underlies neurodegenerative conditions such as Huntington's disease (HD) and Parkinson's disease (PD). Autophagy clears aggregate-prone proteins, and pharmacologic inhibition of the mTOR pathway — which induces autophagy — is beneficial in HD. As complications can arise with long-term mTOR inhibition, identifying mTOR-independent autophagy pathways represents a major therapeutic goal. In Nature Chemical Biology, David Rubinsztein and colleagues now describe a novel autophagy pathway and identify compounds that increase protein aggregate clearance.

A screen for compounds that increased clearance of mutant α-synuclein — a protein linked to PD — uncovered a drug that antagonizes L-type calcium channel activity (verapamil) and a compound that activates G_i signaling pathways to inhibit adenylyl cyclase (clonidine). The L-type calcium channel agonist (±)-Bay K8644 retarded clearance of mutant α-synuclein and huntingtin. These drugs had no effect in autophagy-deficient Atg5^-/- mouse embryonic fibroblasts.

Clonidine reduces cyclic AMP (cAMP) levels, suggesting that cAMP signaling inhibits autophagy. Indeed, stimulation of the cAMP effector Epac blocked clearance of α-synuclein aggregates. Epac fostered Rap2B-phospholipase C (PLCε)-mediated formation of IP₃, which increased intercellular calcium levels. The effect of (±)-Bay K8644 and verapamil on autophagy strongly suggests a central role for calcium signaling in this process.

The protease calpain is upregulated in HD and activated by cAMP and L-type calcium channel signaling. Small interfering RNA against calpain increased clearance of mutant huntingtin and α-synuclein, whereas constitutively active calpain increased aggregation and reduced autophagosome formation. The effects of constitutively active calpain were not inhibited by verapamil or clonidine, supporting a role for calpain downstream of the calcium channel and cAMP–Epac–Rap2B–PLCε–IP₃ pathways. The effect of calpain on autophagy appears to be independent of mTOR, as concomitant inhibition of mTOR and calpain had an additive effect on promoting autophagy.

Calpain-mediated cleavage activates the heterotrimeric G protein α_s subunit (Gα_s), which promotes adenylyl cyclase activity. Activation of Gα_s increased mutant huntingtin aggregation and blocked clearance of mutant α-synuclein in an adenylyl cyclase-dependent manner, whereas depletion of Gα_s decreased aggregation and increased autophagy. Gα_s appears to be the major target of calpain in autophagy, as inhibition of Gα_s had no additional effect in cells treated with calpain inhibitors.

In some neurodegenerative conditions such as HD and PD, increased intracellular calcium stimulates calpain. Calpain cleaves Gα_s, which increases cAMP levels and completes a devastating positive feedback circuit. These data highlight several new points of pharmacological intervention: reducing intracellular calcium levels (verapamil) and inhibiting cAMP formation (clonidine). Indeed, in Drosophila and zebrafish models of HD, verapamil and clonidine blocked aggregate formation. As infectious microbes are also cleared by autophagy, it will be interesting to determine whether these compounds are useful in treating infection.

Emily J. Chenette
Signaling Gateway

Original Reference:
Williams, A. et al.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway
Nature Chemical Biology 4, 295-305 (2008)
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NF-κB in hypoxia: And now for something completely different

The transcription factor NF-κB induces the expression of HIF-1α, providing a link between innate immunity and the hypoxic response.

During bacterial infection, induction of angiogenic factors such as hypoxia-inducible transcription factor-1 (HIF-1α), and immune response factors such as nuclear factor-κB (NF-κB) prevents hypoxia and cell death. Previous studies indicated that IκB kinase (IKK-β) — which regulates NF-κB activity — and HIF-1α were both post-translationally degraded by oxygen-dependent prolyl hydroxylases (PHDs) under normoxic conditions, suggesting a link between hypoxia and the innate immune response. In Nature, Michael Karin and colleagues now provide evidence for a novel intersection between these two pathways, as they show that NF-κB induces HIF-1α transcription in vivo.

The relationship between the NF-κB and HIF-1α pathways was examined in bone marrow-derived macrophages (BMDMs) from IKK-β-deficient mice. These BMDMs had reduced basal levels of Hif1α mRNA, and HIF-1α and its target genes were not induced during hypoxia or bacterial infection. When evaluated in vivo, hypoxia induced Hif1α mRNA in the livers of wild-type, but not IKK-β-deficient mice. Depletion of IKK-β also correlated with a reduction in both HIF-1α protein and vascular endothelial growth factor (Vegf) mRNA.

One possible explanation for this observation is that NF-κB is required for Hif1α transcription. Indeed, hypoxia promoted nuclear accumulation of the NF-κB RelA subunit and subsequent accumulation of HIF-1α in wild-type macrophages, but not IKK-β-deficient BMDMs. Furthermore, RelA bound directly to the Hif1α promoter. RelA was required for Hif1α induction in fibroblasts under normoxic conditions, suggesting that NF-κB also facilitates basal Hif1α transcription.

Lipopolysaccharide (LPS)-mediated induction of NF-κB in hypoxic cells induced Hif1α promoter activity, leading to accumulation of HIF-1α protein and increased Vegf expression. In normoxic cells, however, LPS treatment caused accumulation of Hif1α mRNA, but not protein, which indicates that accumulation of HIF-1α protein in hypoxic conditions is nonetheless dependent on the concomitant inhibition of PHDs.

Previous studies suggested that HIF-1α induction during hypoxia is predominately achieved through post-transcriptional mechanisms — namely, through the inhibition of PHD-mediated degradation. Karin and colleagues now reveal that hypoxia also stimulates NF-κB-mediated transcription of Hif1α mRNA in vivo. It is interesting to note that NF-κB is necessary for Hif1α transcription in normal and hypoxic conditions. However, post-transcriptional regulation of HIF-1α protein is essential for the hypoxic response, as HIF-1α protein was found to accumulate only in hypoxic conditions. Together, these data document a new role for IKK-β–NF-κB signaling in the response to bacterial infection and hypoxia, as it not only induces cytokines and antimicrobial peptides, but also upregulates pro-angiogenic factors to maintain tissue homeostasis.

Emily J. Chenette
Signaling Gateway

Original Reference:
Rius J. et al.
NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α
Nature 453, 807-811 (2008).
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Oncogenic transformation: Invasion of the microvesicles

Glioma cells that express the oncogenic receptor EGFRvIII form microvesicles that fuse with neighboring, non-transformed cells to promote their oncogenic transformation.

A significant proportion of human gliomas express a truncated, constitutively active mutant of the epidermal growth factor receptor (EGFR) known as EGFR variant III (EGFRvIII). However, only a subset of the transformed cells within the EGFRvIII-positive glioma actually contains a mutated EGFRvIII gene. In Nature Cell Biology, Al-Nedawi et al. now show that EGFRvIII-positive glioma cells shed microvesicles containing EGFRvIII. These microvesicles fuse with benign neighboring cells to promote their oncogenic transformation.

Microvesicles are small membranous vesicles that can contain lipid raft-associated proteins, such as transmembrane receptor kinases. Overexpression of EGFRvIII in cultured U373 glioma cells potentiated EGFRvIII-positive microvesicle formation in vitro. Glioma cells expressing EGFRvIII formed tumors in mice, which shed EGFRvIII-containing microvesicles into the circulatory system.

Microvesicles fuse with the membranes of neighboring cells in a phosphatidylserine-dependent manner. Parental U373 cells that were incubated with EGFRvIII-containing microvesicles showed surface expression of EGFRvIII within 24 hours. Microvesicle transfer caused a 2-fold increase in anchorage-independent growth — a hallmark of oncogenic transformation. EGFRvIII uptake increased Erk1/2 and Akt phosphorylation, and Erk activation was blocked by pharmacologic inhibition of EGFR. Furthermore, annexin V treatment, which inhibits microvesicle fusion, blocked phosphorylation of Erk and Akt, suggesting that microvesicle-mediated transfer of EGFRvIII causes oncogenic transformation by upregulating MAPK signaling.

Activation of canonical EGFR signaling pathways by fusion of EGFRvIII-containing microvesicles resulted in the increased production of pro-tumorigenic factors such as vascular endothelial growth factor (VEGF) and Bcl-x_L, and decreased expression of the cyclin-dependent kinase inhibitor p27/Kip1. Inhibition of vesicle fusion by annexin V blocked these effects, further supporting the importance of EGFRvIII-positive microvesicle fusion in promoting the oncogenic transformation of inert glioma cells.

The horizontal transfer of tumorigenic material has been described for apoptotic cancer cells that transfer oncogenic DNA fragments to neighboring non-transformed cells, and for tumor cells that secrete soluble ligands to induce transformation of proximate cells. Al-Nedawi et al. describe a third novel mechanism of paracrine signaling in a tumor environment in which EGFRvIII protein is shuttled to a neighboring cell via an 'oncosome' to promote oncogenic transformation. If this mechanism is found to be important for human tumorigenesis in vivo, it would help explain why glioma cells that lack an EGFRvIII mutation nonetheless contribute to the overall growth of the tumor. Whether this effect is conserved among other receptor kinases with known roles in human tumorigenesis — MET, cKIT, or other EGFR/ErbB family members — remains to be elucidated.

Emily J. Chenette
Signaling Gateway

Original Reference:
Al-Nedawi, K. et al.
Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells
Nature Cell Biology 10, 619-624 (2008)
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