Integrin adhesion signaling: Tumors declare independence

Integrin α_vβ₃ and c-Src interact to promote anchorage-independent growth and tumor cell survival.

Integrin ligation to the extracellular matrix mediates cell adhesion and activates the signaling molecule focal adhesion kinase (FAK), which drives adhesion-dependent cell survival and proliferation. In Nature Medicine, David Cheresh and colleagues now report that unligated integrin α_vβ₃ can mediate anchorage-independent tumor growth and metastatic potential independently of FAK.

Integrin α_vβ₃ is expressed in many aggressive tumor cells and is known to mediate adhesion-dependent bone metastasis in breast and prostate cancer. David Cheresh and colleagues used immunohistochemistry to demonstrate that α_vβ₃ is expressed in matched lymph node metastases from pancreas and breast tumors. Injecting α_vβ₃-positive human pancreatic cancer cells (FG-β₃ cells) into the pancreas of mice resulted in increased primary tumor mass and spontaneous metastasis to the lymph nodes. Conversely, short hairpin RNA (shRNA) knockdown of the endogenous β₃ subunit from Panc-1 human pancreatic cancer cells inhibited tumor growth and metastasis, suggesting a role for α_vβ₃ in tumor progression. FG-β₃ cells did not show altered proliferation or density, but did display a reduction in apoptosis, indicating that α_vβ₃ affects tumor cell survival.

The authors used immunoprecipitation of adherent cells plated on fibronectin to identify a novel interaction between α_vβ₃ and c-Src that requires the β₃ subunit. They then investigated whether the α_vβ₃–c-Src interaction also occurs in non-adherent cell cultures. Immunoblots of FG-β₃ and α_vβ₃-transfected Panc-1 cells in suspension showed levels of c-Src activation similar to those of adherent cells, whereas FAK phosphorylation was inhibited. Moreover, use of a Src kinase inhibitor (SKI) or knockdown of c-Src reduced the formation of soft agar colonies for these cells, indicating that α_vβ₃-induced anchorage-independent survival is dependent on c-Src.

Injecting mice with FG-β₃ cells containing non-functional c-Src resulted in reduced tumor mass and inhibition of metastasis; deleting the c-Src binding site from the β₃ subunit also reduced tumor size. This indicates that α_vβ₃-mediated tumor growth also requires the c-Src–β₃ interaction in vivo.

Immunoblotting revealed that the c-Src substrate CAS was phosphorylated downstream of c-Src in FG-β₃ cells. By using shRNA knockdown and expression of a dominant-negative mutant of CAS, the authors went on to demonstrate that CAS phosphorylation is required for α_vβ₃–c-Src-mediated anchorage-independent growth.

Integrin α_vβ₃ expression and c-Src activation is known to induce tumor cell migration and invasion. The authors show that α_vβ₃ expression increased migration of cells adhering to vitronectin or fibronectin, whereas it had no effect on invasion through Matrigel. Interestingly, c-Src knockdown or SKI treatment did not affect migration of cells expressing α_vβ₃, indicating that the α_vβ₃–c-Src interaction promotes tumor metastasis independently of migration.

This study shows that integrin α_vβ₃ and c-Src interact to increase metastatic potential and tumor cell growth independently of cell adhesion or migration. Further research should shed light on the therapeutic potential of c-Src inhibition in α_vβ₃-expressing tumors.

Iley Ozerlat
Cell Migration Gateway

Reference:
Desgrosellier, J. S. et al.
An integrin α_vβ₃–c-Src oncogenic unit promotes anchorage-independence and tumor progression
Nature Medicine advance online publication, 6 September 2009 (DOI 10.1038/nm.2009)
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p53 responses: Axin' arrest for death

The scaffold protein Axin interacts with either Tip60 or Pirh2 to promote p53-mediated apoptosis or cell-cycle arrest following DNA damage.

The tumor suppressor p53 can initiate either apoptosis or cell-cycle arrest in response to genotoxic stress. Low levels of DNA damage cause arrest, allowing the damage to be repaired, whereas high levels induce apoptosis, removing cells that may have acquired unrepairable oncogenic lesions. However, it is not yet known how the extent of DNA damage is translated into the appropriate p53-mediated response. In Nature Cell Biology, Sheng-Cai Lin and colleagues now report that the scaffold protein Axin has an important role in determining the nature of the p53 response.

Lethal DNA damage stimulates HIPK2-mediated phosphorylation of p53, which has previously been shown to be critical for the induction of apoptosis. Lin and colleagues found that Axin associates with both p53 and HIPK2, stimulating p53 phosphorylation in response to DNA damage. Indeed, Axin^+/- mice exhibited a dramatic increase in tumor formation, as well as lower levels of p53 transactivation, in response to the mutagen DMBA. Furthermore, small interfering (si)RNA-mediated depletion of Axin blocked damage-induced apoptosis, suggesting a role for Axin in the p53-mediated response to lethal DNA damage.

A screen for interacting proteins revealed that Axin binds to the E3 ubiquitin ligase Pirh2 and the acetyltransferase Tip60. Pirh2 inhibited Axin-stimulated phosphorylation of p53 and the induction of apoptosis, and was found to compete with HIPK2 for Axin binding. In contrast, Tip60 displaced Pirh2 from Axin and reversed Pirh2-mediated inhibition of p53 phosphorylation. Intriguingly, sublethal DNA damage stimulated Pirh2–Axin binding, whereas lethal damage induced Axin–HIPK2 complex formation and co-localization of Tip60, Axin and p53 in the nucleus.

The DNA damage response kinases ATM, ATR and Chk1 were required for the Axin–Tip60 interaction following lethal DNA damage. However, given that ATM and ATR are also activated in response to sublethal damage, it will be important to elucidate how ATM and ATR can promote Axin binding to Tip60 only after severe genotoxic stress.

Thus, ATM and ATR-stimulated interactions between Axin, Tip60 and Pirh2 have a critical role in negotiating the p53-mediated response to DNA damage. Sublethal DNA damage induces an interaction between Pirh2 and Axin, which abrogates Axin–HIPK2 binding, curtails HIPK2-mediated p53 phosphorylation, and stimulates cell-cycle arrest. Conversely, severe damage potentiates the formation of a complex containing Axin, Tip60, HIPK2 and p53, resulting in HIPK2-mediated activation of p53 and induction of apoptosis. These findings highlight a new role for Axin at the fulcrum of p53-mediated arrest versus apoptosis cell fate decisions.

Emily J. Chenette
Signaling Gateway

Reference:
Li, Q., Lin, S., Wang, X. et al.
Axin determines cell fate by controlling the p53 activation threshold after DNA damage
Nature Cell Biology 11, 1128-1134 (2009)
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G-protein-coupled receptors: Signaling from the inside

Parathyroid hormone (PTH) elicits sustained cAMP production by stimulating a prolonged association between the PTH receptor, activated G protein and adenylyl cyclase in early endosomes.

The parathyroid hormone receptor type 1 (PTHR) G-protein-coupled receptor (GPCR) can produce divergent downstream effects in response to its two ligands. PTH binding evokes prolonged cyclic AMP (cAMP) production by stimulating G-protein-mediated activation of adenylyl cyclases, whereas binding of the PTH-related peptide (PTHrP) promotes a transient burst of cAMP. However, the basis for these discrete responses has remained unclear. In Nature Chemical Biology, Jean-Pierre Vilardaga and colleagues now show that the activity and internalization of PTHR differs depending on the identity of the bound ligand. PTH–PTHR complexes are rapidly internalized to early endosomes where they promote persistent cAMP production, whereas PTHrP interacts transiently with PTHR at the plasma membrane and elicits a short-lived response.

Fluorescence resonance energy transfer (FRET) analyses revealed that the identity of the PTHR ligand significantly affects the duration of the ligand–receptor complex. PTHrP interacted with the receptor transiently, whereas PTH formed a persistent complex with PTHR. Despite these differences, both ligands activated Gα_s with similar kinetics; however, PTH–PTHR complexes remained associated with Gα_s for much longer than PTHrP–PTHR. These data indicate that PTH and PTHrP evoke differential responses from PTHR, potentially by affecting receptor conformation. PTH binding locks PTHR into an active conformation and thus permits long-term association with and activation of Gα_s, whereas the rapid disassociation of PTHrP from PTHR does not allow persistent Gα_s signaling.

Live-cell imaging revealed that both PTH and PTHrP were internalized from the cell surface. Surprisingly, PTH, but not PTHrP, remained associated with PTHR, Gα_s and adenylyl cyclases at Rab5-positive early endosomes. Treating cells with both PTH and PTHrP produced cAMP, but only PTH caused sustained cAMP production. Thus, the early-endosome complexes that PTH forms with PTHR, Gα_s and adenylyl cyclase are likely to be the source of PTH-mediated sustained cAMP production. These novel findings challenge the paradigm that cAMP production triggered by GPCRs originates exclusively at the plasma membrane.

The authors suggest that, to achieve these disparate effects on G-protein activation and cAMP production, both ligands initially interact with the PTHR extracellular domain in a similar manner, but that subsequent interactions or allosteric changes in the juxtamembrane domain influence the receptor's interaction with G protein. Indeed, the rate of G-protein association and activation was subtly different for PTH and PTHrP. However, the mechanism by which these ligands differentially stimulate G-protein binding and subsequent endocytosis remains to be elucidated.

Emily J. Chenette
Signaling Gateway

Reference:
Ferrandon, S., Feinstein, T. N., et al.
Sustained cyclic AMP production by parathyroid hormone receptor endocytosis
Nature Chemical Biology advance online publication, 23 August 2009 (DOI 10.1038/nchembio.206)
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GTPase activation at the leading edge: Advancing technologies

The use of 'computational multiplexing' and a photoactivatable version of Rac1 has revealed that RhoA is activated concomitantly to cell advancement, that Rac1 and Cdc42 activation is delayed compared to that of RhoA, and that Rac1 inhibits RhoA.

The GTPases RhoA, Rac1 and Cdc42 control the actin cytoskeleton dynamics that provide the force for cell motility. All three GTPases are activated at the cell front and regulate one another; however, their fine spatio-temporal coordination and mutual regulation are not well characterized. Two studies published in Nature now report the development of technologies that allow the activity of Rho GTPases to be precisely controlled and monitored.

Klaus Hahn and colleagues developed a photoactivatable version of Rac1 (PA-Rac1) by fusing residues 404-547 from Avena sativa Phototropin1 to the amino terminus of constitutively active Rac1. The phototropin fragment contains a flavin-binding LOV domain and a helical extension that adopt a closed conformation in the dark, blocking the binding of effectors to Rac1. Upon illumination, the helix unwinds and releases steric inhibition, thus leading to Rac1 activation. This protein fusion has advantages over previous light-controlled systems in that it is fully genetically encoded, uses non-toxic wavelengths (458 nm), is reversible (with 43 sec half-life of dark recovery), and allows the subcellular location of activation to be precisely controlled. This is due to the fact that residual activation does not exceed 7.5% at 10 μm from an irradiated spot.

Irradiating 20 μm spots at the cell edge of mouse embryo fibroblasts (MEFs) stably expressing PA-Rac1 induced localized phosphorylation of the Rac1 effector PAK, actin polymerization and protrusion of the leading edge while the opposite side of the cell retracted. Conversely, irradiating dominant-negative photoactivatable Rac1 led to local retraction and protrusion in other areas, suggesting that localized Rac1 activation or deactivation is sufficient to induce polar movement. Furthermore, Klaus Hahn and colleagues used PA-Rac1 together with a RhoA biosensor to show that Rac1 activation leads to immediate local inhibition of RhoA in vivo.

Gaudenz Danuser and colleagues used two methods to study the temporal and spatial coordination of GTPase activity relative to the movements of the cell edge during constitutive protrusion/retraction cycles (that were not stimulated by external factors). In their 'computational multiplexing' approach, the activity of Cdc42, Rac1 and RhoA was measured in separate experiments. Signals for each of the three GTPase biosensors were measured every 10 sec in 40-80 sampling windows that moved with the leading edge. Edge velocities were also sampled and the data analyzed so that the timing of GTPase activation was coupled with protrusion or retraction. Using these correlation functions, the authors showed that the GTPases are activated over a fixed time interval relative to the dynamics of the leading edge. The correlation analysis was then repeated in sampling windows at various distances from the cell edge to obtain spatial information. Together these analyses showed that RhoA activation occurs at the cell edge synchronously with protrusion, whereas Rac1 and Cdc42 are activated 2 μm behind the edge with a delay of 40 sec. This suggests RhoA might initiate protrusions, and Cdc42 and Rac1 might stabilize them.

In a complementary approach, biosensors for both RhoA and Cdc42 were expressed in a single cell simultaneously to visualize their activation using four-channel imaging. This method, which gives unprecedented spatial and temporal resolution, showed that Cdc42 is activated after RhoA and confirmed the signaling relationships inferred by the computational multiplexing.

Together the studies of Hahn and Danuser groups provide powerful methods that enable the immediate coupling between signaling pathway states and their morphological outputs to be visualized.

Kim Baumann
Cell Migration Gateway

References:
Wu, Y. I. et al.
A genetically encoded photoactivatable Rac controls the motility of living cells
Nature 461, 104-108 (2009)
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Machacek, M. et al.
Coordination of Rho GTPase activities during cell protrusion
Nature 461, 99-103 (2009)
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