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 paper will be accessible via the Research Library.
Anoikis is a type of apoptosis that results from loss of cell-matrix interactions. In cancer, anoikis is thought to act as a physiological barrier to metastasis; thus, resistance to anoikis may facilitate the survival of cancer cells during systemic circulation, aiding the formation of metastases. Douma et al. have now identified the neurotrophic receptor tyrosine kinase (RTK) TrkB as a potent and specific suppressor of caspase-associated anoikis and linked it to the promotion of metastases.
TrkB is a neurotrophic RTK, which, along with its ligand brain-derived neurotropic factor (BDNF), is required for the ontogeny, function and survival of the mammalian nervous system. In a functional screen in rat intestinal epithelial (RIE) cells for genes that conferred resistance to anoikis, TrkB was identified along with oncogenic RasV12.
TrkB was shown to disrupt epithelial cell organization, which was aggravated by ligand co-expression, resulting in the complete loss of cell-cell contact. The authors confirmed that TrkB specifically prevented anoikis induced by disruption of adhesion, and not apoptosis in general.
The authors found that TrkB-expressing epithelial cells stimulated with BDNF generated a signal powerful enough to resist anoikis even in the absence of any other survival factors. TrkB was shown to activate protein kinase B (PKB/AKT), which by itself is sufficient to suppress anoikis. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) upstream of PKB significantly impaired TrkB-mediated survival.
Non-malignant epithelial cells engineered to express TrkB and TrkB/BDNF, rapidly formed tumors when introduced into nude mice. In vivo imaging showed that, in contrast to controls, TrkB-expressing epithelial cells injected into mice metastasized to the lungs and heart; injection of TrkB/BDNF-expressing cells induced an even more pronounced effect. Microscopic pathological analysis showed the capacity of TrkB to induce solid, undifferentiated, non-encapsulated tumors. These tumors were able to efficiently invade both blood and lymphatic vessels in various tissues, including liver, kidney, lung and heart.
TrkB and BDNF are frequently overexpressed in human cancer, and a recent sequence analysis of the tyrosine kinome in colorectal cancers has shown mutations within the kinase domain of TrkB. The results raise the possibility that TrkB activation may represent an early event during multi-step tumorigenesis that contributes to metastasis. Drug-mediated inactivation of TrkB may impact the tumorigeneic and metastatic capacities of tumors caused by mutation or overexpression of TrkB.
Brenda Riley, Assistant Editor Signaling Gateway
article
Sirith Douma, Theo Van Laar, John Zevenhoven, Ralph Meuwissen, Evert Van Garderen & Daniel S. Peeper Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB Nature, 430, 1034 - 1039 (26 August 2004); doi:10.1038/nature02765
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Lance A. Liotta and Elise Kohn Anoikis: Cancer and the homeless cell Nature, 430, 973 - 974 (26 August 2004)
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The transient receptor potential (TRP) superfamily of ion channels act as cellular sensors that translate external signals into changes in membrane excitability and increased intracellular calcium. These ubiquitously expressed cation channels consist of tetramers made up of six-transmembrane polypeptide subunits. Members of the canonical TRP (TRPC) subfamily function as receptor-operated channels, activated by G protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) stimulation. TRPCs display constitutive activity, and although not understood, regulatory mechanisms must exist to prevent continuous Ca2+ influx. Clapham and colleagues now show that growth factor stimulation initiates a response termed 'rapid vesicular insertion of TRP' (RiVIT), whereby TRPC ion channels are rapidly translocated to the cell surface from vesicles held in reserve just under the plasma membrane.
TRPC5 is expressed in the mammalian brain where it forms homomers, which are present in growth cones, and TRPC5/TRPC1 heteromers, which are confined to the cell body and proximal processes. TRPC5 is predominantly localized in the plasma membrane. Fluorescently labeled TRPC5 (TRPC5-EGFP) accumulated within intracellular vesicles, and translocated to the plasma membrane upon epidermal growth factor (EGF) stimulation. A three-fold increase in TRPC5 current indicated the formation of functional TRPC5 homomeric channels, but not heteromeric TRPC5/TRPC1 channels.
TRPC5 membrane insertion coincided with membrane translocation of the Akt kinase and TRPC5 insertion was blocked upon phosphotidylinositide 3-kinase (PI(3)K) inhibition, indicating that PI(3)K is required for the rapid increase in channel availability. The PI(3)K effector Rac1 was also shown to be necessary and sufficient for growth factor-induced TRPC5 plasma membrane insertion. Rac1 directly binds and activates phosphatidylinositol 4-phosphate 5-kinase (PIP(5)Kα) and consequently phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2 is required for membrane ruffling, and may also be involved in vesicular exocytosis; thus the authors suppose it may stimulate TRPC5-vesicle translocation.
Application of the nerve growth factors (NGFs) brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) to rat primary hippocampal neurons transiently increased the amount of surface-accessible TRPC5. This occurred in parallel with Rac1 stimulation, indicating that TRPC5 translocation occurs through a PI(3)K-dependent mechanism in neurons. Calcium is known to control growth cone extension through an unidentified ion channel. PIP(5)Kα expression inversely correlates with neurite length, and TRPC5 functions downstream of PIP(5)Kα in this process.
Although this work provides an insight into the gating and regulation process of TRP channels, many questions are yet to be answered: Do TRPC5-vesicles fully fuse with the membrane? By what mechanism does the TRPC5-dependent cation influx inhibit neurite outgrowth? How does TRPC5 interact with the exocytotic machinery? And finally, is this rapid vesicular incorporation unique to TRPC5?
Brenda Riley, Assistant Editor Signaling Gateway
article
Vassilios J. Bezzerides, I. Scott Ramsey, Suhas Kotecha, Anna Greka & David E. Clapham Rapid vesicular translocation and insertion of TRP channels Nature Cell Biology, 6, 709 - 720 (2004); doi:10.1038/ncb1150
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Genomic instability and subsequent aneuploidy frequently occur in advanced human cancer and are thought to play a causal role in cancer development and progression. What causes genomic instability in cancer cells is unclear. Previous studies suggest that defects in the mitotic spindle checkpoint may be involved, although loss-of-function mutations in spindle checkpoint genes are rarely observed in human cancers. Hernando et al. now show that aneuploidy can arise from defects in the retinoblastoma (Rb) pathway that indirectly deregulates the spindle checkpoint.
The authors found that E1A, a viral oncoprotein that inactivates the Rb pathway, significantly upregulates the expression of the vital spindle checkpoint component MAD2. Cells lacking Rb also displayed increased MAD2 expression. One consequence of inactivation of the Rb pathway is the deregulation of the E2F transcription factors normally kept in check by Rb, prompting the authors to investigate a link between E2F and MAD2 expression. Indeed, the MAD2 promoter has several putative E2F-binding sites and the authors showed in vivo that the MAD2 gene is a direct transcriptional target of E2F.
Many of E2F's target genes are regulated in a cell cycle-specific manner, and this was found to also be the case for Mad2 expression. Mad2 levels began to increase after cells entered S phase and reached a maximum in G2/M. However, similar to other E2F targets, Mad2 exhibited aberrantly high levels of expression throughout the cell cycle in cells lacking functional Rb.
Retinoblastomas, which often exhibit a loss of Rb function and E2F deregulation, also showed high levels of Mad2 expression. The authors observed a clear link between deregulated E2F activity and aberrant Mad2 expression in human neuroblastomas, with Mad2 expression levels closely tied to patient prognosis.
Increased levels of Mad2 may compromise mitotic events, thus predisposing a cell to genomic instability. The authors confirmed that Rb defective cells are frequently aneuploid. Both Rb defective cells and cells overexpressing Mad2 take longer to complete mitosis and have difficulty completing cytokinesis and segregating chromosomes. When Mad2 was reduced to near normal levels in Rb defective cells, the fraction of aneuploid cells was also reduced, showing that although there may be other factors contributing to chromosome instability, aberrant Mad2 expression is vital for this process.
The authors propose that during normal cell proliferation the cell cycle is hardwired to the spindle checkpoint through E2F. When mutations that disrupt Rb function occur, E2F activity is deregulated. This leads to uncontrolled proliferation and aberrant Mad2 expression, which can ultimately result in aneuploidy. Hence, in searching for the reasons underlying genomic instability in cancer, one might be looking in the wrong place when focusing on mutations in proteins that regulate mitosis.
Brenda Riley, Assistant Editor Signaling Gateway
article
Eva Hernando, Zaher Nahle, Gloria Juan, Elena Diaz-Rodriguez, Miguel Alaminos, Michael Hemann, Loren Michel, Vivek Mittal, William Gerald, Robert Benezra, Scott W. Lowe & Carlos Cordon-Cardo Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control Nature, 430, 797 - 802 (12 August 2004); doi:10.1038/nature02820
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Inflammation: Protein A causes invasion of the PMNs
Found on the skin and intranasally in 25-30% of the population,
Staphylococcus aureus is a pathogen that is mostly harmless.
However, infection can lead to pneumonia and sepsis, particularly
in infants and in intensive care units; this is a particularly
acute problem given the increased occurrence of multidrug resistant
strains.
S. aureus infection of the lower respiratory tract induces
an influx of inflammatory cells such as polymorphonuclear leukocytes
(PMNs) as a result of secretion of chemokines and cytokines, including
TNF-α,
by airway epithelia. Although this response is necessary for bacterial
clearance, it can lead to tissue damage and pneumonia. Prince
and colleagues now shed light on the mechanism by which S.
aureus triggers this PMN mobilization.
Bacterial cell wall-associated protein A is expressed in unusually
high quantities on S. aureus strains isolated from patients
with airway infections. Exposure of epithelial cells to purified
protein A in vitro resulted in dose-dependent and TNF-α-
independent induction of the PMN chemokine interleukin-8 (IL-8).
Protein A was necessary and sufficient for the activation of the
kinases p38 and JNK1/2,
resulting in NF-κB stimulation and IL-8 expression.
This typical proinflammatory signaling pathway is shared by TNF-α.
In a screen for the epithelial cell surface receptors of protein
A, the ubiquitous TNF-α receptor TNFR1 was isolated. Protein
A bound to TNFR1 competitively with TNF-α in vitro and
in vivo. TNF-α signaling results in the mobilization
of TNFR1 to the cell surface, where it sheds into the airway lumen.
Accordingly, after exposure to protein A, TNFR1 shedding into
culture media was observed independently of TNF-α, while
protein A-null S. aureus failed to elicit this response.
Like TNF-α, protein A induced recruitment of the death domain
protein TRADD
and TNF-receptor-associated factor 2 (TRAF2)
to TNFR1, and thus a functional requirement for TRAF2 in protein
A signaling was demonstrated.
The finding that protein A signaling can mimic TNF-α signaling
in lung inflammatory responses was confirmed in vivo: mice
intranasally infected with protein A-null S. aureus showed
a significantly lower incidence of pneumonia than mice infected
with wild-type S. aureus. Further, mice deficient in TNFR1
infected with the wild-type staphylococci also showed reduced
incidence of pneumonia, suggesting that the protein A-TNFR1 interaction
aggravates the disease.
This study shows that protein A signaling through TNFR1 is the
chief pathway for S. aureus induced pneumonia. An accompanying
News and Views article places these findings in the context of
the complex TNF-α-associated signaling events, and cautions
that full comprehension of signaling by this inflammatory cytokine
in vivo is called for before TNF-α and its receptors
can be targeted in the treatment of inflammatory disorders.
Julia Howlett, Assistant Editor Signaling Gateway
article
Marisa I Gómez, Aram Lee, Bharat Reddy, Amanda Muir,
Grace Soong, Allyson Pitt, Ambrose Cheung & Alice Prince Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nature Medicine, 10, 842 - 848 (2004); doi:10.1038/nm1079
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Birgitta Henriques Normark, Staffan Normark & Anna Norrby-Teglund Staphylococcal protein A inflames the lungs. Nature Medicine, 10, 780 - 781 (2004); doi:10.1038/nm0804-780
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