Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the research highlight will be accessible via the Updates page.
Rgs5-mediated inhibition of G-protein signaling results in a more mature tumor vasculature, which increases the effectiveness of T-cell immunotherapy in tumors.
Tumor vasculature is less organized and more fragile than that of normal tissue. The altered vessel architecture is chiefly caused by abnormal pericytes and endothelial cells, and hinders the infiltration of immune cells into the tumor. Little is known about why these pericytes and endothelial cells are abnormal. Hamzah et al. now report in Nature that vascular abnormalities present in mouse pancreatic tumors are caused by Rgs5 (regulator of G-protein signaling 5), which is a known GTPase-activating marker for pericyte progenitor cells.
Hamzah et al. noted that the life span of RIP1–Tag5 mice — which develop pancreatic tumors expressing defined T-antigens (TAGs) — was reduced when the mice lacked Rgs5 (RIP1–Tag5 X Rgs5-/- mice). This appeared to be related to the fact that RIP1–Tag5 X Rgs5-/- mice displayed more normal vessel architecture, which led to both better oxygen supply to tumors and reduced vascular permeability. Cytological examination of the RIP1–Tag5 X Rgs5-/- vessels showed a more mature pericyte phenotype as indicated by expression of αSMA (α-smooth muscle actin) and NG2. The connection between Rgs5 and vessel stability was also demonstrated by the finding that Rgs5-/- mice had less brain edema after induction of cerebral ischemia than wild-type mice.
But how could Rgs inhibition be used therapeutically if tumors appear to thrive in Rgs5-/- mice? Hamzah et al. primed CD4+ and CD8+ T cells ex vivo against TAG epitopes from RIP1–Tag5 X Rgs5-/- tumors. After these were transferred back into the mice, the authors observed an increase in the presence of T cells in the tumor parenchyme, which led to a strong anti-tumor immune response. This indicated that the more normal vessel architecture of RIP1–Tag5 X Rgs5-/- mice allowed T cells to infiltrate the tumors more readily than in RIP1–Tag5 mice. Indeed, the medium life span of RIP1–Tag5 X Rgs5-/- mice receiving primed T cells was increased by one third.
The current study bears some similarity to work showing that VEGF (vascular endothelial growth factor) inhibition initially leads to a more normal vessel architecture due to increased pericyte density, but at the same time it confers higher drug susceptibility to the tumors. An anti-G-protein signaling approach as presented here may prevent the development of therapeutic resistance caused by the expression of other angiogenic factors. Furthermore, the fact that Rgs5 is expressed in many tumors encourages a more detailed exploration of the mechanism and the pharmacologic potential of anti-G-protein signaling-based strategies.
Mirko von Elstermann Functional Glycomics Gateway
Original Reference:
Hamzah J. et al. Vascular normalization in Rgs5-deficient tumours promotes immune destruction Nature453, 410-414 (2008) Full text | PDF | Subscribe to Nature
Tumor necrosis factor (TNF) stimulation primes macrophages for higher inflammatory responses through an interferon-response factor 1 (IRF1-) and interferon β- (IFN-β-)mediated autocrine loop similar to that used by toll-like receptors (TLRs).
Macrophages produce TNF and can cause chronic inflammatory diseases such as rheumatoid arthritis upon prolonged exposure to the cytokine. Although it is known that this response requires the activation of transcription factors such as nuclear factor-κB (NF-κB), the long-term effects of TNF stimulation are yet to be elucidated. The immediate effects of TNF exposure overlap with the TLR-dependent response toward pathogens, but it is unknown whether TNF causes the sustained IFN-β production that is a long-term effect of TLR signaling. IFN-β production is induced by activation of IRF following TLR ligand binding. IFN-β docks to its own receptor, leading to the transcription of other IRFs and STATs (signal transducers and activators of transcription), which re-initiate IFN-β and induce IFN-α production, thus creating an autocrine loop. In Nature Immunology, Yarilina et al. now report that TNF signaling has a long-term effect on IFN-β production.
The authors observed a sustained change in gene expression for 48 hours in TNF-stimulated human macrophages. Interestingly, the interferon-response genes STAT1 and IRF7 were expressed as a late response to TNF stimulation, suggesting that, similar to TLR signaling, TNF may induce IFN-β production. The expression of late-response genes was also significantly reduced in IFN-β receptor-deficient macrophages and levels of IFN-β messenger RNA (mRNA) increased after stimulation of the macrophages with TNF. Macrophages from mice lacking IRF1 exhibited reduced expression of late-response genes after TNF stimulation compared with those of wild-type mice. The authors conclude that IRF1 mediates TNF-dependent IFN-β production, which is then propagated by both an autocrine loop, and the expression of STAT1 and further interferon-response genes.
Yarilina et al. noted that mice lacking IRF1 were less susceptible to endotoxic shock, suggesting that IFN-β may boost the effects of TNF. TNF stimulation prior to the binding of virus-derived molecules to TLR increased IFN-β production, pointing to a role for TNF-induced IFN-β expression in anti-viral responses. Furthermore, IFN-β production and expression of target genes was upregulated in macrophages from patients with rheumatoid arthritis, suggesting that this new pathway of TNF signaling plays a major role in inflammatory autoimmune conditions.
The results of Yarilina et al. demonstrate how TNF and TLR signaling converge in an autocrine loop of IFN-β production, which is initiated and sustained through the expression of interferon-response genes. This loop enhances anti-viral responses but, if persistently activated, can also lead to chronic inflammation. Thus, this study may help in understanding the pathogenesis of rheumatoid arthritis, as well as highlighting the necessity of understanding how single stimuli are able to drive the long term effects of signal transduction.
Mirko von Elstermann Functional Glycomics Gateway
Original Reference:
Yarilina, A., Park-Min, K. H., Antoniv, T., Hu, X. & Ivashkiv, L. B. TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes Nat. Immunol.9, 378-387 (2008) Full text | PDF | Subscribe to Nature Immunology
Aging: Notching up stem cell differentiation
A mutant form of lamin A activates the Notch signaling pathway to promote precocious mesenchymal stem cell differentiation.
Lamin proteins line the interior of the nucleus and contribute to both chromatin structure and gene regulation. A mutant version of lamin A called progerin is known to cause the premature-aging disease Hutchinson–Gilford Progeria Syndrome (HGPS). Constitutive expression of progerin produces nuclear defects and increases DNA damage, although the mechanism of action behind the associated accelerated aging has remained unclear. In Nature Cell Biology, Scaffidi and Misteli now show that progerin promotes premature differentiation of mesenchymal stem cells by upregulating the Notch signaling pathway.
Genome-wide microarray analysis revealed that progerin expression correlated with the induction of Notch pathway proteins in immortalized skin fibroblasts. Whereas cell lines derived from HGPS patients express high levels of progerin and Notch effectors, the amount of Notch intracellular domain (NICD) protein — the active version of Notch that functions as a transcriptional co-activator — remained constant in HGPS cells. This indicates that proteins downstream of NICD may mediate the effect of progerin. Indeed, the Notch target genes' co-activator SKIP (Ski-interacting protein) was upregulated in HGPS cells. Interestingly, SKIP was no longer associated with the nuclear lamina in HGPS cells, suggesting that progerin interferes with normal SKIP localization and permits unregulated SKIP signaling.
Bone and adipose tissue originate from mesenchymal progenitor cells and exhibit striking alterations in HGPS patients. Expression of progerin in immortalized human mesenchymal stem cells (hMSCs) not only upregulated Notch pathway effectors, it also caused the misregulation of differentiation markers such as collagen IV and osteopontin in undifferentiated cells. In addition, when lineage-specific differentiation was induced, progerin expression enhanced osteogenesis, but inhibited differentiation of hMSCs into adipose tissue — a hallmark of HGPS. Overexpression of wild-type lamin A protein, however, caused an intermediate phenotype. Expression of NICD in hMSCs similarly induced osteogenic differentiation and blocked adipogenesis, suggesting that the Notch pathway may be involved in HGPS. Indeed, pharmacologic inhibition of Notch signaling partially suppressed the effect of progerin expression in hMSCs.
This study shows that progerin activates the Notch pathway, resulting in an abnormal MSC differentiation that may underlie the premature aging of HGPS patients. As normal cells also express low levels of progerin, determining whether progerin plays a role in normal tissue homeostasis to promote terminal differentiation and aging will be important.
Emily J. Chenette Signaling Gateway
Original Reference:
Scaffidi, P. & Misteli, T. Lamin A-dependent misregulation of adult stem cells associated with accelerated ageing Nature Cell Biology10, 452-459 (2008) Full text | PDF | Subscribe to Nature Cell Biology
Tumorigenesis: eNOS is enough
Oncogenic KRas initiates and maintains tumor growth by stimulating the PI3K–AKT–eNOS signaling pathway, which activates wild-type HRas and NRas.
Human cancers frequently contain mutations in Ras GTPase family members. The phosphoinositide 3-kinase (PI3K)–AKT effector pathway is necessary to maintain Ras-mediated tumorigenesis, but the signaling events downstream of PI3K and AKT that are critical for tumor maintenance remain to be defined. In Nature, Kian-Huat Lim, Brooke B. Ancrile, David F. Kashatus and Christopher M. Counter now report that AKT-mediated activation of endothelial nitric oxide synthase (eNOS) maintains tumor growth resulting from oncogenic KRas by promoting the activation of other Ras family members.
Counter and colleagues examined the contribution of various PI3K effectors in tumor maintenance, and found that small hairpin RNA-mediated inhibition of the known AKT substrate eNOS blocked tumorigenesis in PI3K-transformed cells. AKT phosphorylates eNOS on serine 1117, and expression of the non-phosphorylatable mutant eNOSS1117A did not rescue eNOS deficiency. Therefore, eNOS is an important effector of PI3K-mediated tumorigenesis.
eNOS catalyzes nitric oxide synthesis, which promotes Ras nitrosylation and GTP binding. Constitutive PI3K signaling stimulated nitrosylation and GTP loading of endogenous NRas and HRas in KRas-deficient cells. Ras activation was blocked by pharmacologic inhibition of PI3K or depletion of eNOS. In vivo, eNOS depletion inhibited the tumorigenesis of HRas-transformed cells, which was rescued by eNOS, but not eNOSS1117A. Furthermore, a chemical carcinogen that specifically elicits oncogenic HRas mutations caused significantly fewer tumors in eNOS-/- mice than their wild-type counterparts.
Whereas eNOS clearly affects H- and NRas activity, KRas mutations prevail in human cancers. Over 90% of human pancreatic carcinomas contain mutationally activated KRas, and active eNOS was detected in both human pancreatic cancer cell lines and tumor samples. Depletion of eNOS significantly reduced the size and growth rate of tumor cells in vitro and blocked growth of established tumors in vivo.
eNOS stimulates Ras activity by facilitating GTP loading; therefore, eNOS should not affect mutationally activated and constitutively GTP-bound KRas. Why, then, does loss of eNOS block KRas-mediated tumor growth? Interestingly, depletion of endogenous, wild-type HRas and NRas in pancreatic cancer cells restricted tumor growth. Re-introduction of HRas or NRas, but not nitrosylation-deficient HRas or NRas, rescued growth, suggesting that eNOS-mediated nitrosylation and activation of endogenous HRas and NRas is critical for the growth and maintenance of KRas-mutant tumors.
These data describe the existence of a positive-feedback circuit whereby oncogenic KRas activates PI3K and AKT to promote eNOS phosphorylation, which in turn stimulates formation of active HRas and NRas to maintain tumor growth. Pharmacologic inhibition of eNOS was previously shown to block tumor progression, and this study suggests a biological mechanism for this effect. Whether this pathway is maintained in tumors derived from mutant NRas, HRas or other Ras family members will be interesting to determine.
Emily J. Chenette Signaling Gateway
Original Reference:
Lim, K.-H., Ancrile, B. B., Kashatus, D. F & Counter, C. M. Tumour maintenance is mediated by eNOS Nature452, 646-649 (2008) Full text | PDF | Subscribe to Nature
