Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Bax-and Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells

Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist to Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the anti-apoptotic activity of AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and PI K inhibitors PD98059 and wortmannin, suggesting the involvement of a 3 PKC-dependent, MAPKand PI K-independent survival pathway. The PKC inhibitor rottlerin restores apoptosis induced by serum 3 deprivation. In addition, phosphorylation of PKC and PKC/, but not of PKC/ , increases in serum-starved H358 cells and in H322 cells II treated with AR/IGF1 combination and is blocked by calphostin C. Combination of AR and IGF1 increases p90 and Bad Rsk phosphorylation as well as it inhibits the conformational change of Bax by a PKC-dependent mechanism. Finally, PKC, PKC or p90Rsk siRNAs block the anti-apoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90 restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90 -dependent pathway, Rsk Rsk which leads to Bad and Bax inactivation. This signalling pathway is different to that used by single factor. MESH


Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Baxand Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells
In non small cell lung cancer (NSCLC), growth factors that bind to tyrosine kinase receptors, have been characterized for their ability to inhibit apoptosis and promote cell survival through autocrine/paracrine loops ( ). They represent promising targets for new therapeutic 1 strategies as illustrated by the clinical development of epidermal growth factor (EGF) receptor signaling pathway inhibitors and they might be an alternative to conventional chemotherapy which fails to improve survival in most patients with NSCLC. A significant percentage of tumorigenic human lung epithelial cells overexpress amphiregulin (AR) and EGF-related cytokines ( ) as well as members of the erbB receptor 2 family, in particular the EGF receptor (erbB-1/HER1) that binds AR, and erbB-2/neu (HER2) ( , ). Such overexpression has been associated 3 4 with shortened survival in patients with NSCLC ( ). Type 1 insulin like-growth factor (IGF1) receptor is also overexpressed in NSCLC ( ); 3- 6 7 it is a major survival factor that protects cells from apoptosis induced by number of stimulus ( ), including serum withdrawal ( , ). Moreover, 8 9 10 we have previously shown, on two NSCLC cell lines H322 and H358 that 1) H358 cells secrete AR and IGF1 that cooperate to inhibit apoptosis induced by serum deprivation in an autocrine manner ( ). Those two factors also protect H322 cells from apoptosis in a paracrine 11 manner when cells are incubated with conditioned medium from H358 cells (CM) or with the two growth factors at concentrations found in the H358 CM ( ). 2) AR inhibits apoptosis through an IGF1-dependent survival pathway, independently of the EGF receptor ( ). 3) H358 cells 11 11 resist to Bax-induced apoptosis through an inactivation of Bax conformational change ( ). 12 The IGF1 receptor is known to exert its anti-apoptotic function mainly through activation of the phosphatidylinositol 3-kinase (PI K)/Akt 3 pathway ( ) whereas the EGF receptor usually activates the Ras/Raf/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated 13 kinase (ERK) pathway ( ) and the PI K pathway ( ). These two growth factor receptors signaling pathways promote NSCLC cells survival ( 14 3 15 , ) and converge with the cell death machinery to the phosphorylation and inactivation of Bad, a pro-apoptotic member of the Bcl2 family 16 20 21 Furthermore, mitochondrial membrane-based protein kinase A (PKA) and protein kinase C (PKC) could also be implicated in Bad phosphorylation. PKA is able to phosphorylate Bad on Ser ( , , ) and Ser ( ). Finally, the MAPK-activated p90 ribosomal S6 kinase 112  Based on our previous published results, the aim of the present study was first to characterize the molecular cascade activated by both factors AR and IGF1, acting in cooperation, to protect NSCLC cells H322 and H358 from apoptosis induced by serum deprivation; secondly to see whether or not this molecular cascade leads to Bax inactivation. We show here that combination of AR and IGF1 stimulates a PKC -and ζ PKC -dependent, MAPK-and PI K-independent, survival pathway. PKC inhibit serum deprivation-induced apoptosis through activation of δ 3 p90 and inactivation of the pro-apoptotic molecules Bax and Bad. Finally, the pathways activated by AR or IGF1, used as single agent, are Rsk different from that observed, here, with the two factors used in combination.

EXPERIMENTAL PROCEDURES Cell culture, serum withdrawal and H358 conditioned medium
The human H358 and H322 cells were cultured in RPMI 1640 medium (Gibco, Cergy Pontoise, France) with 10 heat-inactivated fetal % calf serum. For serum deprivation, cells were rinsed twice in PBS and cultured in serum-free medium. After 8h, serum-free medium was changed and cells cultured as described in each experimental condition. Conditioned medium (CM) from H358 cells was collected 72 h after serum-free culture, centrifuged (5000g) at 4 C and stored at 80 C until use.°−°W e have previously shown that in H358 CM obtained from serum-free medium cell culture, IGF1 level was about 1 ng/ml, as usually observed in CM of other analyzed cancer cell lines, but the AR level was very high: 5 ng/ml, 50 more than in CM from other analyzed cancer cell lines ( ). In all experiments performed in this manuscript, levels of AR and IGF1 in H358 CM were controlled as described ( ) and 11 11 shown to be similar. Transient transfections were carried out using Fug ne 6 (Roche Diagnostics, Meylan, France) according to the manufacturer s protocol.

Quantification of apoptotic cell death
Cells were incubated with the different effectors diluted in the medium for 96 h at the concentration indicated. The morphological changes related to apoptosis were assessed by fluorescence microscopy after Hoechst 33342 (5 g/ml, Sigma-Aldrich) staining of cells and the percent μ of apoptotic cells was scored after counting at least 500 cells. The proteolytic activation of pro-caspase-3 was evaluated by Western Blotting.

Immunofluorescence staining
Cells cultured on Lab-Tek were transfected as indicated. 96h after transfection, cells were fixed in 20 C acetone for 5 min. After

RESULTS
We have previously demonstrated that H358 cells resisted to serum-deprivation apoptosis and that serum-free conditioned medium (CM) from H358 cells protected H322 NSCLC cells from apoptosis, although they are normally sensitive to serum deprivation ( ). The 11 anti-apoptotic activity of H358 CM was completely abolished by a pre-incubation with the anti-AR neutralizing antibody and to a lower extent (about 50 ) by the anti-IGF1 antibody. In contrast, neutralization of other putative growth factors did not alter anti-apoptotic activity of H358 % CM ( ). In addition, AR and IGF1 recombinant proteins, used in combination at the concentrations detected in H358 CM, mimicked the 11 anti-apoptotic activity of H358 CM, whereas AR or IGF1, used as a single agent, partially inhibited apoptosis ( ). We have taken advantage of 11 H358 and H322 NSCLC cells properties to analyze here the apoptosis inhibition pathway promoted by AR and IGF1. 3 The PI K, MAPK as well as the PKC and PKA are the prevalent intracellular pathways transmitting receptor-mediated anti-apoptotic 3 signals from the cell surface to the nucleus. In order to determine which of these pathways was responsible of the apoptosis inhibition, specific inhibitors ( ) were added to H322 cells cultured in H358 CM (with characterized levels of AR and IGF1 as described in methods) or in table 1 presence of AR and IGF1 recombinant proteins at the same concentrations as in H358 CM ( ). figure 1 Incubation of serum-starved H322 cells in the presence of the p42/44 MAPK inhibitor PD98059 and the PI K inhibitor wortmannin 3 enhanced their apoptosis but did not affect the protective effect of the H358 CM or of the combination of AR and IGF1 ( ). figure 1A, B, F Similarly, the anti-apoptotic activity of the H358 CM did not change with U0126, another p42/44 MAPK inhibitor, SB202190 or SB203580, two p38 MAPK inhibitors, and the PI K inhibitor LY294002 (data not shown). In contrast, low concentrations of calphostin C (200 nM) and 3 staurosporine (10 nM), which both inhibit the PKC pathway, restored apoptosis of H322 cells cultured in H358 CM or in presence of AR and IGF1, but did not modify the percentage of apoptosis induced by serum deprivation in H322 cells ( ). The PKA inhibitor H89 figure 1C, D, F also enhanced apoptosis of H322 cells cultured in serum-free condition and of H358 CM but without statistical significance ( ). In figure 1E addition, dibutyryl 3 , 5 -cyclic adenosine monophosphate (dbcAMP), which activates PKA, did not inhibit serum deprivation-induced ′ ′

apoptosis (
). PD98059 and wortmannin, but not calphostin C, blocked the partial anti-apoptotic activity of AR alone, used at the same figure 1E concentration as in H358 CM, whereas wortmannin, but not PD98059 or calphostin C, seemed prevent the partial inhibition of apoptosis observed with single agent IGF1 ( ). figure 1F Similar experiments performed on H358 cells also demonstrated that PKC inhibitors staurosporine and calphostin C sensitized H358 cells to serum deprivation-induced apoptosis, while wortmannin and PD98059 had no significant effect ( ). figure 2 Taken together, these data suggested that combination of AR and IGF1 present in the H358 CM inhibited apoptosis through activation of a PKC-dependent survival pathway in NSCLC cells but not p42/p44 and p38 MAPK nor PI K pathways. AR or IGF1 single agent did not 3 activated such PKC-dependent survival pathway.

H358 CM and AR/IGF1 combination involve atypical PKC and novel PKC to inhibit apoptosis
Among the PKC family, several PKC isoforms could be involved. The specific classical PKC inhibitor Go6976, the calcium chelator BAPTA-AM (data not shown) or the inhibitor of both classical and novel PKC basindolylmaleimide I (GF109203X) did not influence the capacity of H358 CM to inhibit the apoptosis induced by serum deprivation in H322 cells ( ). The phorbol 12-myristate 13-acetate figure 3A, C (PMA), which activates PKC, mimicked the anti-apoptotic effect of H358 CM in serum-starved H322 cells and this effect was totally block by GF109203X, demonstrating that GF109203X was active ( ). Surprisingly, 1 M of rottlerin, which inhibits novel PKC , restored figure 3D μ δ apoptosis of H322 cells cultured in H358 CM ( ). Altogether, these data suggested that apoptosis inhibition involved the atypical PKC figure 3B and the novel PKC but was independent of classical PKC and of the known anti-apoptotic effect of PMA.
δ In order to control that the PKC-dependent survival pathway was independent of the MAPK pathway, p42/p44 MAPK phosphorylation status was examined in H358 and H322 cells using specific antibody recognizing Thr /Tyr phosphorylated residues. As expected, in both 202 204 cell lines, PD98059 incubation dramatically prevented p42/p44 phosphorylation ( ). In serum-starved H358 cells, p42/p44 MAPK figure 3E phosphorylation level did not change as compared to control culture condition. Similar levels of phosphorylated p42/p44 were also detected in the presence of specific PKC inhibitors. In H322 cells, as compared to serum-starved condition, H358 CM enhanced the level of phosphorylation of p42/p44 MAPK, but this effect was not abolished by specific inhibitors of PKC calphostin C, staurosporine or rottlerin ( ). This suggested that the PKC-dependent anti-apoptotic pathway induced by H358 CM did not involve p42/p44 MAPK, even if this figure 3E latter was activated by H358 CM.  ). figure 4B This suggested that the cooperation of AR and IGF1 in H358 CM, but not AR or IGF1 single factor, inhibited serum deprivation-mediated apoptosis through PKC and PKC / activation. of Bad Ser was not detected (data not shown). 136 Taken together, these results indicated that the combination of AR and IGF1, but not AR or IGF1 used alone, inhibited serum deprivation-mediated apoptosis a PKC-dependent pathway involving activation of p90 and inactivation of Bad through phosphorylation. via

PKC-dependent survival pathway, activated by AR and IGF1, prevents Bax conformational change
Previous studies have shown that the Bax protein changed of conformation and exposed its N terminus domain during apoptosis ( , , ).
12 34 35 Using an epitope-specific antibody that only recognizes the N terminal extremity of Bax when it is exposed, we showed that serum deprivation increased Bax conformational activation in H322 cells but not in H358 cells ( ). H358 CM or combination of AR and IGF1 recombinant figure 6 proteins prevented Bax conformational-activation; the level of fluorescence, reflecting Bax conformational change, was similar in H322 cells treated with H358 CM or with combination of AR and IGF1 and in untreated control cells ( ). AR or IGF1 used alone did not have the figure 6B same effect as the combination of the both growth factors. The presence of the specific PKC inhibitor calphostin C in H358 CM or in serum-free medium supplemented with AR and IGF1, enhanced Bax activation and restored the level of Bax N terminus staining to the level of serum-starved H322 cells. Similarly, calphostin C enhanced the staining of Bax N terminus in serum-starved H358 cells ( ). figure 6A These observations highly suggested that inhibition of apoptosis by combination of AR and IGF1 came from the inhibition of Bax conformational change by a PKC-dependent mechanism.

AR/IGF1 combination inhibits apoptosis through a PKC-, PKC-and p90 -dependent pathway Rsk
Taken together, our results suggested that H358 CM and combination of AR and IGF1 inhibited apoptosis-induced by serum deprivation through a PKC-and p90 -dependent pathway. This pathway led to inactivation of Bad as well as conformational inactivation of Bax.

Rsk
In order to confirm the involvement of PKC and p90 , we analyzed the effect of silencing subtype-specific PKC and p90 by siRNA in IGF1, but not in presence of AR or IGF1 alone ( ). figure 7D Altogether, these data suggested that the inhibition of apoptosis by H358 CM or by the combination of AR and IGF1 involved both PKCζ and PKC as well as p90 , whereas single factor AR or IGF1 acted through different pathway. δ Rsk In order to verify the activity of PKC and p90 on serum deprivation-mediated apoptosis, H322 cells were transiently transfected with Rsk mutant constructs and apoptosis of transfected cells, detected by immunofluorescence, was analyzed ( ). Overexpression of figure 8 constitutively active PKC or PKC isoforms strongly inhibited apoptosis of serum-starved H322 cells, as compared to GFP-transfected cells (

DISCUSSION
In this manuscript, we have shown that, in two NSCLC cell lines H358 and H322, AR and IGF1 cooperate to prevent apoptosis induced by serum deprivation by activating a PKC-dependent survival pathway different to that used by single factor AR or IGF1. Atypical PKC , novel ζ PKC and p90 are the principal mediators in this signal transduction. This survival pathway leads ultimately to Bad as well as Bax δ Rsk inactivation.
We have previously identified that AR and IGF1 are the key anti-apoptotic molecules in the conditioned medium from H358 cells and that the cooperation of those growth factors inhibits apoptosis both IGF1 and EGF receptors in NSCLC ( ). IGF1 receptor exerts its via 11 anti-apoptotic effect essentially through activation of the PI K pathway ( ), which promotes cell survival by phosphorylating Bad ( ). In 3 13 18-21 agreement, the IGF1-mediated inhibition of apoptosis in our model of NSCLC cells seems prevented by PI K inhibitors ( ). However, 3 figure 9A alternative pathways have been proposed such as the MAPK pathway or the activation of Raf-1 and its translocation to the mitochondria ( , 36 37 ). PKC are also involved in IGF1-dependent pathway activated during mitogenesis ( , ), cell migration ( ), proliferation ( ) or 38 39 40 AR-mediated inhibition of apoptosis ( ). To date, PKC pathway has not been described to directly propagate the EGF receptor figure 9A signaling. However, EGF receptor triggers PKC activity ( ) at least in part downstream of phospholipase signaling ( ) and EGF has recently 46 47 been shown to induce fibroblast contractility and motility a PKC -dependent pathway ( ). In our model of NSCLC cells, the via δ 48 anti-apoptotic activity of H358 CM, mediated by AR/IGF1 cooperation, is prevented by PKC inhibitors but not by PI K or MAPK inhibitors, 3 demonstrating the activation of an original PKC-dependent survival pathway ( ). Of note, although PKC might activate the MAPK figure 9B pathway ( , , , ), the phosphorylation of ERK1/2 proteins, by serum deprivation in H358 cells and by H358 CM in H322 cells, is 25 26 49 50 independent of PKC; this result confirms that MAPK activation is not involved in the apoptosis inhibition mediated by combination of AR and IGF1 in our model. Similarly, H89 restores apoptosis but the PKA activator dbcAMP has no significant protective effect, suggesting that the PKA pathway is also not involved in cells survival.
Specific PKC isoforms may be either anti-or pro-apoptotic, depending on the nature of the apoptotic stimuli and of the cell type ( ). Caspases can cause limited proteolysis of PKC , generating a 40 kDa C-terminal fragment. It was proposed that the catalytic fragment of δ PKC is pro-apoptotic and the holoenzyme is anti-apoptotic ( , ). In our model, we do not detect the 40 kDa fragment, except after PMA δ 54 56 treatment (data not shown). In addition, the novel PKC inhibitor GF109203X has no effect on the anti-apoptotic activity of H358 CM, suggesting that the PKC -dependent pathway triggered by AR and IGF1 is independent of the anti-apoptotic activity of phorbol esters.

57
We show here that calphostin C and PKC or PKC siRNA inhibit survival pathway activated by the combination of AR and IGF1 but ζ δ have no effect when each growth factor are used as a single agent. This PKC-dependent survival pathway, activated by the combination of AR and IGF1, is different of the pathways activated by AR or IGF1 used as single agent. Each factor activates the classical ways described respectively for the EGF and IGF1 receptors ( ). Altogether, our results suggested that the cooperation between AR and IGF1 takes figure 9A place upstream of the PKC cascade, probably at the level of receptors. In agreement with this hypothesis, we have previously identified that AR inhibits apoptosis through an IGF1-dependent survival pathway in NSCLC ( ), but the mechanism responsible of such cooperation remains to 11 be determined.
In our study, the PKC-survival pathway involves the activation of p90 and the phosphorylation of Bad on Ser ( ). This is Rsk 112 figure 9B confirmed by AR/IGF1 survival pathway inhibition, when we use siRNA directed against p90 . We also demonstrate that overexpression of a Rsk catalytically inactive form of Rsk prevents the anti-apoptotic activity of H358 CM, demonstrating the major role of this protein in AR/IGF1 survival pathway. Accordingly, the MAPK-activated p90 has been reported to protect cells from Bad-induced apoptosis by phosphorylation ). These observations confirm the non-involvement of the PKA and PI K in the survival pathway induced by AR and IGF1. 21 3 Forced expression of PKC in prostate cancer cells confers a significant resistance to phorbol esters, associated with an inhibition of Bax ε conformational rearrangements ( ). This study suggested that an association of PKC with Bax might neutralize apoptotic signals propagated 31 ε through a mitochondrial death-signaling pathway. We previously showed that H358 NSCLC cells resist to Bax-mediated apoptosis, due to an inhibition of Bax conformational change and of its translocation from the cytosol to the mitochondria ( ). Here, we show that Bax 12 conformational change inhibition is due to AR/IGF1 cooperation and the activation of a PKC-dependent pathway ( ). figure 9B In conclusion, it is well established that growth factors, in particular EGF-related peptides and IGF1 are involved in survival of malignant human lung epithelial cells through activation of intracellular MAPK and/or PI K pathways. Our study has demonstrated for the first time that 3 1) the two growth factors, AR and IGF1, cooperate to induce an original PKC -, PKC -and p90 -dependent survival pathway in NSCLC ζ δ Rsk cells, independently of other pathways classically described for these two growth factors alone.
2) The PKC-dependent pathway induced by AR/IGF1 cooperation can modify Bad phosphorylation and inactivate Bax conformational change. We thus have identified a new survival pathway mediated by AR/IGF1 cooperation that might contribute to lung tumor development and progression. Further studies on the relevance of this pathway on fresh tumor samples will allow development of new therapeutic strategies. in vivo

Ackowledgements:
We thank Dr Greenberg for the kind gift of dominant-negative Rsk2 construct (plasmid) and Dr Baier-Bitterlich for providing the expression plasmid containing the constitutively active form of PKC and PKC . We thank Carole Niang for technical assistance.