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.
Lysophosphatidic acid (LPA) controls actin dynamics indirectly, through cell surface receptor-mediated signaling, and directly, by binding and inhibiting villin.
Rearrangement of the actin cytoskeleton is crucial for the control of cell morphology and migration. Actin-binding proteins regulate the polymerization of actin into filaments, and the cross-linking of these into bundles. LPA is known to induce cytoskeletal rearrangements, but the mechanisms involved are not well understood. Now, two studies in the Journal of Biological Chemistry show how LPA can influence actin dynamics in different ways. First, Masayuki Masu and colleagues report that in mouse embryos, extracellular LPA controls phosphorylation of an actin binding protein through a cell surface receptor and Rho GTPase signaling cascade, leading to the formation of specialized lysosomes. Meanwhile, Seema Khurana and colleagues used in vitro methods to show that LPA and phosphatidylinositol 4,5-bisphosphate (PIP2) compete for binding sites in villin, an actin binding protein, with opposing effects on actin reorganization.
Extracellular LPA (see the Lipid of the month) is produced by the secreted enzyme autotaxin, which is encoded by the Enpp2 gene. Defects including failures in yolk sac angiogenesis kill Enpp2-/- mouse embryos before birth, but the cellular mechanisms involved are not well understood. Masu and colleagues used a whole embryo culture system to probe the role of autotaxin–LPA in specialized yolk sac cells. These visceral endoderm (VE) cells usually have distinctively large lysosomes, but these were fragmented in VE cells from Enpp2-/- embryos.
Using pharmacological inhibitors and electroporation of dominant-negative constructs, the authors showed that the formation of large lysosomes in VE cells requires LPA receptor signaling, Rho GTPase, the kinase ROCK, and the downstream LIM kinase (LIMK). Cofilin, an actin binding protein that stimulates filament disassembly, is phosphorylated and inactivated by LIMK. The steady-state levels of cofilin phosphorylation were significantly decreased in Enpp2-/- VE cells, and this correlated with a decrease in polymerized actin. Culturing the embryos with compounds to either disrupt or stabilize actin filaments confirmed that actin polymerization is involved in VE lysosome formation, and that dynamic regulation of actin turnover is required. Without autotaxin–LPA signaling to regulate cofilin, the balance of actin polymerization is lost and the lysosomal defects ensue.
Another actin-binding protein, villin, is specific to endothelial cells, where it regulates actin dynamics, cell morphology and migration. PIP2 is known to bind villin and modify its actin regulatory functions. Khurana and colleagues found that LPA also binds villin in vitro, and kinetic analysis revealed that villin has a slightly higher affinity for LPA than for PIP2. On binding, neither lipid induced global changes in the conformation of full-length villin, but each had a different effect on the secondary structure of villin peptides. This difference had functional consequences for full-length villin; c-Src kinase was unable to phosphorylate the recombinant protein whether PIP2 was present or absent, but it was able to do so in the presence of LPA. In vitro, actin filaments are cross-linked into bundles by villin, but this did not occur in the presence of LPA. LPA also inhibited the actin capping function of villin as well as its depolymerizing activity. PIP2, by contrast, only inhibited the last function.
Whereas the study by Masu and colleagues identifies a pathway by which extracellular LPA modifies the actin cytoskeleton, in vivo binding of villin would involve intracellular LPA. The different cellular membranes have distinct phospholipid compositions, suggesting that LPA or PIP2 binding might regulate villin localization as well as phosphorylation. Intracellular LPA production involves phospholipase enzymes, not autotaxin, and is more tightly regulated than extracellular production, although it is not certain that the two LPA pools are entirely separate. Nevertheless, control of actin dynamics from inside or outside the cell could be critical for many of the pathological functions of LPA.
References:
Koike, S., Keino-Masu, K., Ohto, T., Sugiyama, F., Takahashi, S. & Masu, M. Autotaxin/lysophospholipase D-mediated LPA signaling is required to form distinctive large lysosomes in the visceral endoderm cells of the mouse yolk Sac J. Biol. Chem. published online 5 October 2009 (DOI: 10.1074/jbc.M109.012716) Abstract | PDF
Tomar, A., George, S. P., Mathew, S. & Khurana, S. Differential effects of lysophosphatidic acid and phosphatidylinositol 4,5-bisphosphate on actin dynamics by direct association with the actin-binding protein villin J. Biol. Chem. published online 5 October 2009 (DOI: 10.1074/jbc.C109.060830) Abstract | PDF
Two studies reveal a crucial role for NF-κB signaling in cancers that possess activating mutations in KRAS.
Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide. Small-molecule inhibitors that target epidermal growth factor receptor (EGFR) have shown some clinical success; however, mutations in KRAS, which are detected in 20-30% of NSCLC adenocarcinomas, render these therapeutics mostly ineffective. Two reports in Nature now demonstrate that nuclear factor-κB (NF-κB) signaling is essential for the survival of cancer cells with mutations in KRAS, revealing a potential new pathway for therapeutic intervention.
In addition to mutations in KRAS, loss of p53 activity is a frequent event in NSCLCs. Constitutively active KRAS-G12D was previously shown to stimulate the NF-κB pathway, whereas wild-type p53 antagonizes NF-κB activity. Jacks and colleagues found that localization of the NF-κB subunit p65 (also known as RELA) in mouse embryonic fibroblasts was not affected by either expression of KRAS-G12D or loss of p53. However, expression of KRAS-G12D and concomitant loss of p53 caused p65 to accumulate in the nucleus.
Tumor cells from mice that expressed KRAS-G12D and lacked p53 (KP mice) exhibited high levels of NF-κB DNA-binding activity; similar observations were made with human NSCLC cell lines. Blocking NF-κB pathway activation through the expression of a dominant-negative mutant of NF-κB inhibitor-α (IκBα; also known as NFKBIA), or knockdown of either p65 or the NF-κB pathway protein NEMO (also known as IKBKG), induced apoptosis in KP cells, but not wild-type cells. These data reveal that the canonical NF-κB pathway is important for the survival of lung cancers with mutations in KRAS and TP53 (which encodes p53). Indeed, the dominant-negative IκBα mutant blocked tumor formation and attenuated the growth of established tumors in KP mice.
A crucial role for NF-κB in cancers that express mutant KRAS was also observed by Hahn and colleagues. The authors found that TANK-binding kinase 1 (TBK1; a non-canonical IκB kinase) was required for the survival of human cancer cells that express mutant KRAS, as suppression of TBK1 induced apoptosis in these cells. Consistent with previous observations, the selective inhibition of the Ras effector RALB also induced death in KRAS-mutant cells.
Gene expression analyses revealed that the KRAS-mutant lung cancers show evidence of Ras and NF-κB pathway activation. Indeed, the levels of NFKBIA and the NF-κB precursor NFKB1 were reduced in KRAS-mutant cells, which were restored by the suppression of TBK1. Additional experiments found that mutant KRAS and TBK1 were required for the nuclear accumulation of the NF-κB subunit REL, as well as the expression of the anti-apoptotic protein BCL-XL. Therefore, oncogenic KRAS activates RALB-TBK1 signaling to induce activation of NF-κB and promote cancer cell survival.
Together, the studies from Jacks and colleagues and Hahn and colleagues suggest that the inhibition of the NF-κB pathway might be an effective strategy for treating lung adenocarcinomas that possess mutations in KRAS and p53, as well as other cancers that express constitutively active KRAS.
Emily J. Chenette Signaling Gateway
Reference:
Meylan, E. et al. Requirement for NF-κB signaling in a mouse model of lung adenocarcinoma Nature462, 104-107 (5 November 2009) Full text | PDF | Subscribe to Nature
Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1 Nature462, 108-112 (2009) Full text | PDF | Subscribe to Nature
Cancer biology: The benefit of being single
TGFβ signaling drives single cell motility and stimulates hematogenic metastasis in breast cancer cells.
Regulation of cell motility and invasion is important for the dissemination of tumor cells from their primary location to lymph or blood vessels during metastasis. The epithelial to mesenchymal transition of cancer cells leads to increased cell motility and tumor progression, and is known to involve transforming growth factor β (TGFβ). In Nature Cell Biology, Erik Sahai and colleagues now report that the transient and local activation of TGFβ signaling in breast cancer cells switches them from cohesive movement to single cell motility and promotes hematogenous metastasis.
Using intravital imaging of mammary carcinoma cells, the authors show that only 5% of primary tumor cells were motile and that they moved either singly or collectively. Interestingly, motile behavior was not maintained in lymph node metastases. Imaging of fluorescent reporter genes demonstrated that TGFβ is active primarily in single cells and that this correlates with the nuclear localization of Smad2 and Smad3, which are phosphorylated by TGFβ and form a complex with Smad4 that accumulates in the nucleus. Importantly, the increase in TGFβ activity was not maintained in lymph node and lung metastases, suggesting that TGFβ is only activated transiently.
So, how does the activity of TGFβ affect the mode of tumor cell migration? When cultured in the presence of TGFβ, tumor cells moved singly instead of growing in colonies. This was inhibited by knockdown of Smad4, suggesting a role for TGFβ-mediated transcription in determining the mode of migration. Using microarray analysis, Sahai and colleagues identified several genes that are upregulated in the cells treated with TGFβ. Knockdown studies revealed that these genes have distinct roles in the switch from collective to single cell motility, suggesting that TGFβ activates a programmed transcriptional mechanism to influence cell motility.
Intravital imaging also revealed that expression of a 'dominant-negative' TGFβ receptor in rat breast cancer cells triggered a switch back to cohesive movement, whereas overexpression of TGFβ promoted single cell motility. This suggests that TGFβ is necessary for single cell motility in vivo. But how does this switch in cell motility affect metastasis?
The authors confirmed that cells lacking TGFβ signaling were moving collectively and could only enter lymphatic vessels to disseminate into the lymph nodes. Conversely, cells with permanently hyperactive TGFβ signaling entered the blood efficiently, but were ineffective at forming lung metastases as prolonged TGFβ signaling inhibited growth. Thus, the transient activation of TGFβ enables single cells to enter the blood and its subsequent inactivation permits growth at secondary sites.
This study demonstrates that dynamic TGFβ signaling regulates both the mode of migration and the metastatic route of breast cancer cells. Future studies will hopefully further elucidate the distinct mechanisms for lymphatic and hematogenic metastasis.
References:
Giampieri, S. et al. Localised and reversible TGFβ signaling switches breast cancer cells from cohesive to single cell motility. Nature Cell Biology11, 1287-1296 (2009) Full text | PDF | Subscribe to Nature Cell Biology
DNA damage: Keeping telomerase at bay
Activation of the DNA-damage response pathway elicits phosphorylation of Pif1, which restrains the activity of telomerase at double-stranded DNA breaks.
The enzyme telomerase maintains chromosome integrity by synthesizing telomeres at chromosome ends. What prevents telomerase from adding telomeres to double-stranded DNA breaks (DSBs)? In Nature Cell Biology, Svetlana Makovets and Elizabeth Blackburn now report that DNA damage signaling induces phosphorylation of the telomerase inhibitor Pif1 (petite integration frequency 1). Phosphorylation of Pif1 blocks the activity of telomerase at DNA breaks but not at chromosome ends.
In budding yeast, DSBs sustained during normal growth or as a result of genotoxic stress activate the kinases Tel1 and Mec1 — orthologs of mammalian ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related), respectively — to induce phosphorylation of the checkpoint kinases Chk1 and Rad53. Makovets and Blackburn found that Pif1, which is known to antagonize telomerase function at DNA ends, is phosphorylated following DNA damage. Deletion of MEC1 or RAD53 blocks DNA damage-induced Pif1 phosphorylation, although Rad53 is not required for the recruitment of Pif1 to DSBs. These data indicate that the DNA damage response pathway regulates Pif1 phosphorylation but not Pif1 localization.
Intriguingly, expression of a Pif1 mutant that cannot be phosphorylated at Thr 763, Ser 765, Ser 766 or Ser 769 (pif1-4A) permits the erroneous addition of telomeres at DSBs; the same effect was observed in PIF1-null cells. By contrast, pif1-4A has no effect on telomere addition at natural chromosome ends. Phosphorylation of Pif1 is not induced by stalled replication forks or by nocodazole-mediated mitotic arrest. Thus, Mec1-dependent phosphorylation of Pif1 between residues 763–769 is activated specifically in response to DNA breaks, and this potentiates its ability to inhibit telomerase activity at DSBs.
Additional experiments reveal that DSBs induce Pif1 phosphorylation at Thr 763 and Ser 766, which is essential for the restraint of telomerase function at DSBs. However, it is not yet known how the phosphorylation of Pif1 blocks telomerase activity at these sites. It will also be important to elucidate whether Pif1 is a direct target of Rad53 and Dun1 (a kinase downstream of Rad53), and whether this pathway is conserved in mammalian cells.
Emily J. Chenette Signaling Gateway
References:
Makovets, S. & Blackburn, E. H. DNA damage signalling prevents deleterious telomere addition at DNA breaks Nature Cell Biology11, 1383-1386 (2009) Full text | PDF | Subscribe to Nature Cell Biology
