Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor

Receptor-mediated endocytosis may represent an important mechanism whereby peptide hormones exert their biological effects. The ability of recombinant insulin-like growth factor (IGF)-I to be internalized by cultured cells was evaluated in BRL-3A2 cells, a rat liver-derived cell line which lacks insulin receptors. Since recombinant IGF-I does not bind to the Type II IGF receptor, all specific binding of 125I-IGF-I in BRL-3A2 cells represents binding to the Type I receptor. Exposure of BRL-3A2 cells to IGF-I resulted in a rapid 50% downregulation of Type I IGF receptors. Only one-half of these binding sites were sensitive to treatment with trypsin, a phenomenon which indicates that the peptide and its receptor were internalized after the cells were exposed to IGF-I. In conclusion, these experiments demonstrate that IGF-I can be internalized by cultured cells via the Type I IGF receptor, and suggest that IGF hormone action may be exerted by receptor-mediated endocytosis.     

Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.     

Efficient processing and expression of human nerve growth factor receptors in Xenopus laevis oocytes: effects on maturation.

The nerve growth factor (NGF) receptor is an integral membrane protein that is phosphorylated and heavily glycosylated. Determination of the amino acid sequence by molecular cloning indicates that the receptor is a cysteine-rich protein which contains a signal peptide sequence and spans the lipid bilayer with a single transmembrane sequence. A single mRNA of 3.8 kilobases was observed for the receptor, of which 1.5 kilobases is coding sequence. We have used microinjection of receptor RNA in Xenopus laevis oocytes to obtain cell surface expression of the receptor. The presence of NGF receptors in oocytes was verified by radioimmunoassay, specific binding of [125I]NGF, and metabolic labeling followed by immunoprecipitation. The NGF receptor protein was rapidly processed in oocytes and displayed extensive glycosylation. Furthermore, the presence of NGF receptors in oocytes potentiates the ability of progesterone to induce maturation.     

Molecular kinetics of nerve growth factor receptor trafficking and activation

A growing body of evidence indicates a close relationship between tyrosine kinase receptor trafficking and signaling. Biochemical and molecular analyses of the expression, fate, and kinetics of membrane trafficking of the nerve growth factor (NGF) receptor TrkA were performed in PC12 cells. Pulse-chase experiments indicate that TrkA is synthesized as a 110-kDa N-glycosylated precursor that leads to the mature 140-kDa form of the receptor with a half-life of conversion of approximately 24 +/- 0.5 min. Neuraminidase digestion shows that modification of the carbohydrate moiety of the receptor by sialylation occurs during maturation. The 140-kDa form is rapidly translocated to the cell surface as assessed by cell surface biotinylation performed on intact PC12 cells. Mature receptor half-life is approximately 138 +/- 4 min and is shortened to 86 +/- 8 min by NGF treatment. Flow cytometric analysis indicates that NGF induces clearing of this receptor from the cell surface within minutes of treatment. The addition of NGF decreases the half-life of cell surface gp140(TrkA) from 100 to 35 min and leads to enhanced lysosomal degradation of the receptor. The process of NGF-induced TrkA internalization is clearly affected by interfering with ligand binding to p75(NTR). An analysis of receptor activation kinetics also shows that receptor signaling primarily takes place from an intracellular location. Together, these data show that the primary effect of NGF treatment is a p75(NTR)-modulated decrease in TrkA transit time at the cell surface.     

Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor.

Neurotrophin-3 (NT-3) has low-affinity (Kd = 8 x 10(-10) M), as well as high-affinity receptors (Kd = 1.8 x 10(-11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), NT-3 also binds to the low-affinity NGF receptor, a molecule that we suggest to designate low-affinity neurotrophin receptor (LANR). NT-3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT-3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT-3 to its high-affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand-fold higher than those corresponding to their Kd to their high-affinity neuronal receptors. Thus, specific high-affinity NT-3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed.     

Madin-Darby canine kidney cells display type I and type II insulin-like growth factor (IGF) receptors

Using affinity cross-linking techniques, we report the presence of type I IGF and type II IGF receptors in Madin-Darby canine kidney cells, a line of cells lacking insulin receptors. The IGF receptors were further characterized by competition binding studies and found to be similar to IGF receptors in other tissue types. In Madin-Darby canine kidney cells, the type I IGF receptor binds IGF-I greater than IGF-II greater than insulin and the type II IGF receptor binds IGF-II and IGF-I with approximately the same affinity, but does not bind insulin.     

Binding of brain-derived neurotrophic factor to the nerve growth factor receptor

The neurotrophic proteins BDNF and NGF are related in their primary structures, and both have high- and low-affinity receptors on their responsive neurons. In this study, we investigate the extent to which these receptors can discriminate between BDNF and NGF. We found that a 1000-fold excess of the heterologous ligand is needed to reduce binding to the high-affinity receptor by 50%, but that the same concentrations of BDNF and NGF similarly reduce the binding of either ligand to the low-affinity receptor. Results obtained with cells transfected with the low-affinity NGF receptor gene indicate that these cells bind BDNF, in addition to NGF, whereas cells before transfection do not. These data indicate that the low-affinity NGF receptor is also a low-affinity BDNF receptor and that whatever is conferring high-affinity binding and biological response also considerably reinforces the ability of the low-affinity receptor to discriminate between NGF and BDNF.     

A serological assay for the detection of cell surface receptors of nerve growth factor

When single-cell suspensions prepared from embroyonic day 8 (E8) chick sensory ganglia are incubated with nerve growth factor (NGF), anti-NGF antiserum, and complement, an NGF-dependent cytotoxic kill of 20 (±3)% of the ganglia cells is observed. This percentage is increased by a factor of two when only the neuronal cells are tested. No kill is observed on the nonneuronal cell population representing 50% of the ganglia dissociate. When E8 sensory ganglia cells are cultured in the presence of NGF following cytotoxic kill, the large, phase-bright NGF-reponsive neurons are missing from the culture. These results indicate that the cells recognized in the cytotoxicity assay have to carry NGF-binding sites of type I, which is the one with the higher affinity of the two types of NGF-binding sites (I and II) present on sensory ganglia cells. This conclusion is further supported by the following data: (a) half maximal cytotoxicity is reached already at a concentration of NGF which is below the K_D of binding site I; (b) a washing step which removes all NGF bound to type II receptors while leaving a high percentage of type I receptors occupied has no effect on the percentage of ganglia cells killed. Using the cytotoxicity assay the presence of high-affinity binding sites of type I can be demonstrated on sensory ganglia cells from E8 chick embryos but not from E4 embryos and not on liver and heart cells from E8 embryos. Further, type I receptor-bearing cells were detectable in the brain using this assay. At E8, NGF receptors could be detected on cells of the forebrain and the tectum but not on brain stem cells. Cytotoxic kill of forebrain cells was found to be especially high at E8 and E9, and decreased by E10.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.     

The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.     

A comparison of nerve growth factor binding protocols with native and mutant PC12 cells

A comparison has been made of various methods for measuring binding of nerve growth factor (NGF) to PC12 cells in suspension, on plates, and by a combination of the two. Results indicated that the extensive washing in the plate binding assay removed some cell surface ligand, underestimated the fast receptor binding, and overestimated the proportion of internalized ligand. In addition, the binding and internalization by a nonresponding PC12 mutant cell line has been studied. The nonresponding mutants had fewer total NGF receptors (10–50%) than normal cells in any binding assay. However, when measured in the suspension assay, the mutant cells showed both fast and slow binding receptors, in proportion approximately equivalent to those found on native PC12 cells. The PC12 nonresponders in suspension were also found to internalize and degrade low levels of NGF, in proportion to their reduced receptor number. Different results concerning PC12 wild type and mutant cells that have been reported in the literature may be due to the particular binding assay protocol that was used.     

Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA- induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N- acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.     

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.     

Nerve growth factor (NGF) receptor on rat glial cell lines. Evidence for NGF internalization via p75NGFR.

Two types of nerve growth factor (NGF) receptors have been described: high affinity (class I) and low affinity (class II). Biological responses to NGF are thought to be mediated by class I receptors, whereas the role of class II receptors is less clear. While some neuronal cells express both receptor types, only class II receptors have been detected on glial cells. Two glial cell lines, peripheral Schwannoma D6P2T and central 33B glioma cells, were employed to investigate the properties of class II receptors in the absence of class I receptors. These cell lines were found to express NGF receptors identified as class II by a low nanomolar dissociation constant, rapid dissociation kinetics at 4 degrees C, and trypsin sensitivity. The receptor was found to bind brain-derived neurotrophic factor with similar affinity as NGF. The responsible binding molecule appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a heterogeneously glycosylated protein of 60-80 kDa with a tendency to aggregate. All receptor bands affinity-labeled with radioiodinated NGF were immunoprecipitated with anti-p75NGFR antibody, but not with anti-p140prototrk antiserum. In these cells, which express p75NGFR as only NGF receptor, a time- and temperature-dependent appearance of a nondisplaceable, trypsin-resistant, acid wash-stable ligand fraction, followed by an increase of trichloroacetic acid-soluble radiolabel in the medium was observed. This sequestration resembled receptor-mediated internalization with subsequent degradation of NGF. Whether this ligand processing indicates a functional role of p75NGFR in glial cells remains to be shown.     

Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza- adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)- dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF- dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.     

Regulation of amphiphysin1 by mitogen-activated protein kinase: its significance in nerve growth factor receptor-mediated endocytosis

Amphiphysin1, which can simultaneously bind to dynamin1 and the clathrin adaptor AP-2, is essential for dynamin1 recruitment during receptor-mediated endocytosis, but little is known about its regulatory mechanism. Here, we purified a 120-kDa mitogen-activated protein kinase (MAPK) substrate protein from porcine brains and identified the protein as amphiphysin1. Serine phosphorylation of amphiphysin1 was rapidly induced by nerve growth factor (NGF) in PC12 cells, and the induction was blocked by a MAPK inhibitor. Furthermore, when phosphorylated by MAPK in vitro or by NGF treatment in vivo, amphiphysin1 failed to bind to AP-2, but its association with dynamin1 was unaffected. Consistent with this, mutation of consensus MAPK phosphorylation sites increased amphiphysin1 binding to AP-2 and their intracellular colocalization. Thus, we propose that MAPK phosphorylation of amphiphysin1 controls NGF receptor/TrkA-mediated endocytosis by terminating the amphiphysin1-AP-2 interaction. This perhaps helps to regulate the availability of amphiphysin1-dynamin1 complexes for binding to the endocytic vesicle.     

Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.     

Loss of alpha 2-macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides

We have found that certain naphthalenesulfonamides [e.g., N-6(-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell-surface receptors for alpha 2-macroglobulin, and epidermal growth factor (EGF) in fibroblasts. The loss of alpha 2-macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for alpha 2-macroglobulin, and the EGF receptors do not reappear at the surface when W-7 is removed. Receptor loss was measured as a change in the capacity for binding iodinated ligands; no change in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W-7 did not induce a loss of cell surface beta 2-microglobulin, a membrane protein which is excluded from coated pits and which is not internalized, indicating that the effect of W-7 was specific for membrane receptors and not a result of bulk depletion of plasma membrane. The loss of alpha 2-macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W-7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsible for the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: 1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; 2) the microinjection of calmodulin into cells did not reverse the effects of W-7; using pressure microinjection, we introduced up to a 100-fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rhodamine-labeled alpha 2-macroglobulin, we saw that the W-7 induced inhibition of receptor-mediated endocytosis was the same in injected and uninjected cells; 3) we injected calcineurin, a calmodulin-binding protein, into cells (1-3 pg/cell) and observed no effect on the receptor-mediated endocytosis of rhodamine-labeled alpha 2-macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.     

The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells

Insulin-like growth factors I and II (IGF-I and II) and insulin are chemotactic agents for the human melanoma cell line A2058. As shown in this report, the motility receptor mediating this response is the heterodimeric type I IGF receptor. These three factors are able to compete with 125I-labeled IGF-I for binding to the cell surface with IC50 values equal to approximately 2 (IGF-I), approximately 150 (IGF-II), and approximately 300 nM (insulin). Cross-linking of 125I-IGF-I to the cell surface with disuccinimidyl suberate followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveals a 130-kDa protein (reduced) consistent with the alpha component of a type I receptor and a 38-kDa protein which does not bind insulin, and thus could be another IGF-I cell surface binding protein. The anti-IGF-I receptor monoclonal antibody (alpha IR-3) also competes with labeled IGF-I in binding experiments. In contrast, a control monoclonal antibody, matched to alpha IR-3 with respect to IgG subclass, has no significant effect on IGF-I binding. While alpha IR-3 inhibits the motility induced by IGF-I, IGF-II, and insulin, pertussis toxin (0.01-1.0 micrograms/ml) has no significant effect on the motility induced by the insulin-like growth factors or insulin on this cell line. Therefore, the type I IGF receptor appears to mediate a highly potent pertussis toxin-insensitive motility response to IGF-I, IGF-II, and insulin. In contrast, motility induced by the autocrine motility factor, a cytokine produced by the A2058 cells, is not affected by alpha IR-3 but is extremely sensitive to pertussis toxin. When mixtures of autocrine motility factor and IGF-I are employed to induce chemotaxis, the resulting motility is greater than that induced by either agent alone. These data indicate that motility in this melanoma cell line can be initiated through multiple receptors that stimulate the cells by separate transduction pathways. This capability to respond to multiple stimuli could enhance the metastatic potential.