HER-2/neu Blocks Tumor Necrosis Factor-induced Apoptosis via the Akt/NF-κB Pathway

Overexpression of HER-2/neucorrelates with poor survival of breast and ovarian cancer patients and induces resistance to tumor necrosis factor (TNF), which causes cancer cells to escape from host immune defenses. The mechanism ofHER-2/neu-induced TNF resistance is unknown. Here we report that HER-2/neu activates Akt and NF-κB without extracellular stimulation. Blocking of the Akt pathway by a dominant-negative Akt sensitizes theHER-2/neu-overexpressing cells to TNF-induced apoptosis and inhibites IκB kinases, IκB phosphorylation, and NF-κB activation. Our results suggested that HER-2/neu constitutively activates the Akt/NF-κB anti-apoptotic cascade to confer resistance to TNF on cancer cells and reduce host defenses against neoplasia. Overexpression of HER-2/neucorrelates with poor survival of breast and ovarian cancer patients and induces resistance to tumor necrosis factor (TNF), which causes cancer cells to escape from host immune defenses. The mechanism ofHER-2/neu-induced TNF resistance is unknown. Here we report that HER-2/neu activates Akt and NF-κB without extracellular stimulation. Blocking of the Akt pathway by a dominant-negative Akt sensitizes theHER-2/neu-overexpressing cells to TNF-induced apoptosis and inhibites IκB kinases, IκB phosphorylation, and NF-κB activation. Our results suggested that HER-2/neu constitutively activates the Akt/NF-κB anti-apoptotic cascade to confer resistance to TNF on cancer cells and reduce host defenses against neoplasia. tumor necrosis factor epidermal growth factor IκB kinase hemagglutinin luciferase phosphate-buffered saline dominant-negative Akt phosphorylated Akt estrogen receptor nuclear factor-κB polyacrylamide gel electrophoresis phosphatidylinositol 3-kinase NIH3T3 cells Overexpression of the HER-2/neu (ErbB2) oncogene correlates with poor prognosis in breast and ovarian cancer patients because it enhances the metastatic potential of cancer cells and induces resistance to Taxol and TNF1 (1.Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Science. 1987; 235: 177-182Crossref PubMed Scopus (9851) Google Scholar, 2.Hung M.-C. Zhang X. Yan D.H. Zhang H.Z. He G.P. Zhang T.Q. Shi D.R. Cancer Lett. 1992; 61: 95-103Crossref PubMed Scopus (76) Google Scholar, 3.Yu D. Jing T. Liu B. Yao J. Tan M. McDonnell T.J. Hung M.-C. Mol. Cell. 1998; 2: 581-591Abstract Full Text Full Text PDF PubMed Google Scholar, 4.Hudziak R.M. Lewis G.D. Shalaby M.R. Eessalu T.E. Aggarwal B.B. Ullrich A. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5102-5106Crossref PubMed Scopus (122) Google Scholar, 5.Lichtenstein A. Berenson J. Gera J.F. Waldburger K. Martinez-Maza O. Berek J. Cancer Res. 1990; 50: 7364-7370PubMed Google Scholar). Cancer cells that overexpress HER-2/neu are therefore an excellent target for the development of anticancer therapies. For instance, an anti-HER-2/neu antibody (HerceptinTM) has been used clinically as a potent growth inhibitor of such breast cancer cells (6.Pegram M.D. Lipton A. Hayes D.F. Weber B.L. Baselga J.M. Tripathy D. Baly D. Baughman S.A. Twaddell T. Glaspy J.A. Slamon D.J. J. Clin. Oncol. 1998; 16: 2659-2671Crossref PubMed Scopus (974) Google Scholar), and previous research has shown that overexpression ofHER-2/neu up-regulates p21 Waf1 and leads to resistance by these cancer cells to Taxol (3.Yu D. Jing T. Liu B. Yao J. Tan M. McDonnell T.J. Hung M.-C. Mol. Cell. 1998; 2: 581-591Abstract Full Text Full Text PDF PubMed Google Scholar). Still, the mechanism ofHER-2/neu-mediated TNF resistance in cancer cells remains unclear. The HER-2/neu gene encodes a 185-kDa transmembrane receptor tyrosine kinase with homology to members of the EGF receptor family. Unlike the other EGF receptors,HER-2/neu has an intrinsic tyrosine kinase activity that activates receptor-mediated signal transduction in the absence of ligand. Although EGF can bind to EGF receptor to induce receptor dimerization and activate phosphatidylinositol 3-kinase (PI3K) (7.Hu P. Margolis B. Skolnik E.Y. Lammers R. Ullrich A. Schlessinger J. Mol. Cell. Biol. 1992; 12: 981-990Crossref PubMed Scopus (246) Google Scholar), it is not known whether HER-2/neu homodimer can activate the PI3K pathway without extracellular stimulation. Activation of PI3K generates PtdIns-3,4-P2, which in turn recruits and activates a downstream serine/threonine kinase, Akt. Activated Akt phosphorylates specific targets such as Bad (8.Peso L.D. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1981) Google Scholar), pro-caspase-9 (9.Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2722) Google Scholar), and transcription factor FKHRL1 (10.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar, 11.Kops G.J.P.L. de Reiter N.D. De Vries-smits A.M.M. Powell D.R. Bos J.L. Burgering B.M.Th.B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (949) Google Scholar), with the result of promoting cell survival. Thus, the Akt signaling pathway has a critical role in anti-apoptosis that may contribute to the pathogenesis of cancer (12.Frank T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1519) Google Scholar,13.Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1182) Google Scholar). In this study, we examined the activation of Akt in breast tumor specimens and breast cancer cell lines for its anti-apoptotic roles in HER-2/neu-overexpressing breast cancer cells. We found that Akt was constitutively activated inHER-2/neu-overexpressing breast cancer cells and that Akt activity was required for these cells resistance to TNF-induced apoptosis. We showed that HER-2/neu-overexpressing cancer cells became sensitive to apoptosis when the Akt pathway was blocked by the dominant-negative Akt. Furthermore, we found that Akt activity was required for the activation of both IKK-α and -β, for IκB phosphorylation, and for NF-κB activation. Our results provide a molecular explanation for the finding thatHER-2/neu-overexpressing breast cancer cells are more resistant to TNF-induced apoptosis, leading to poor prognosis and shortened survival of patients. All breast cancer cell lines and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium/F12 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum.HER-2/neu-transformed NIH3T3 cells were generated by transfecting the cells with membrane point-mutated humanHER-2/neu cDNA. Transformed cells were cloned from the transformed foci in three rounds of selection. The DN-Akt transfectants in MDA-MB453 and HER-2/neu-transformed 3T3 cells were established by transfecting these cells with HA-tagged Akt (K179M) cDNA. The transfectants were grown under the same conditions, except that 600 μg/ml of G418 was added to the culture medium. Cells treated with or without TNF were collected at the time interval as indicated and washed once with ice-cold PBS, and apoptosis was analyzed by either a flow cytometry assay or DNA fragmentation, as described previously (14.Shao R. Karunagaran D. Zhou B.P. Li K. Lo S.-S. Deng J. Chiao P.J. Hung M.-C. J. Biol. Chem. 1997; 272: 32739-32742Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15.Shao R. Hu M.C.-T. Zhou B.P. Lin S.-Y. Chaio P.J. von Lindern R.H. Spohn B. Hung M.-C. J. Biol. Chem. 1999; 274: 21495-21498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cell nuclear extracts from samples treated with or without TNF for 30 min were prepared as described previously (14.Shao R. Karunagaran D. Zhou B.P. Li K. Lo S.-S. Deng J. Chiao P.J. Hung M.-C. J. Biol. Chem. 1997; 272: 32739-32742Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15.Shao R. Hu M.C.-T. Zhou B.P. Lin S.-Y. Chaio P.J. von Lindern R.H. Spohn B. Hung M.-C. J. Biol. Chem. 1999; 274: 21495-21498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The nuclear extract (5 μg) was incubated with 1 μg of poly(dI-dC) (Amersham Pharmacia Biotech) on ice for 20 min, and a 32P-labeled double-stranded oligonucleotide containing the κB site of the human immunodeficiency virus was added. Binding of the probe was carried out at room temperature for 20 min. The resulting complexes were resolved in 4% nondenaturing polyacrylamide gel. Cells were washed twice with PBS, scraped into 500 μl of lysis buffer, and incubated on ice for 20 min. After centrifugation at 14,000 × g for 10 min, 500 μg of each supernatant was preincubated with 2 μg of rabbit immunoglobulin G and 50 μl of protein G for 1 h at 4 °C. Endogenous IKK-α was immunoprecipitated overnight with 2 μg of anti-IKK-α antibody (Santa Cruz) and 50 μl of protein G. The immunocomplex was washed five times with lysis buffer, dissolved in loading buffer, and subjected to SDS-PAGE. The protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20 and incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's instructions. The immunoblots were visualized by an enhanced chemiluminescence (ECL) kit obtained from Amersham Pharmacia Biotech. Cell extracts were prepared from samples treated with or without TNF, and immunocomplex kinase assays were performed as described previously (15.Shao R. Hu M.C.-T. Zhou B.P. Lin S.-Y. Chaio P.J. von Lindern R.H. Spohn B. Hung M.-C. J. Biol. Chem. 1999; 274: 21495-21498Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Approximately 0.2 × 106 cells of either MDA-MB453 or its DN-Akt transfectants were cotransfected in 6-well plates with pcDNA3-lacZ and either wild-type NF-κB luciferase (κB-luc) or mutant NF-κB luciferase (mut/κB-luc). After 40 h of transfection, TNF was added to the culture medium as indicated, and both TNF-treated and untreated cultures were continued to incubate for another 8 h. The luciferase activity of each sample was measured with the luciferase assay kit (Promega) and normalized with a β-galactosidase assay. Because overexpression of HER-2/neu induced resistance to TNF (4.Hudziak R.M. Lewis G.D. Shalaby M.R. Eessalu T.E. Aggarwal B.B. Ullrich A. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5102-5106Crossref PubMed Scopus (122) Google Scholar, 5.Lichtenstein A. Berenson J. Gera J.F. Waldburger K. Martinez-Maza O. Berek J. Cancer Res. 1990; 50: 7364-7370PubMed Google Scholar), and the Akt pathway is known to enhance cell survival, we examined whether expression of HER-2/neu correlated with activation of Akt in breast cancers. We compared the levels of activated Akt (phosphorylated Akt) (p-Akt) of 10HER-2/neu-positive and 10 HER-2/neu-negative human breast tumors by immunostaining them with an antibody specific to p-Akt. Although no p-Akt signal was detected in the 10HER-2/neu-negative tumors, 7 of 10HER-2/neu-positive tumors showed strong p-Akt staining, suggesting that expression of HER-2/neu correlates significantly with Akt activation (p < 0.01). As control, all samples were Akt-positive when they were stained with an anti-Akt antibody. Representative stainings of p-Akt are shown in Fig.1 A. To confirm our observation of a correlation between HER-2/neu expression and Akt activation in the clinical samples, we used Western blotting with an anti-p-Akt antibody to analyze p-Akt in nine breast cancer cell lines that showed various expression levels of HER-2/neu. The level of p-Akt paralleled the cell's HER-2/neu expression (Fig. 1 B), indicating that activation of Akt correlates well with expression of HER-2/neu in breast cancer cells. Moreover, this correlation remained the same in the absence of serum, suggesting that activation of Akt corresponds to the level ofHER-2/neu, independent of stimulation of growth factors or cytokines in the serum (i.e. constitutive activation). To create a model system, and to rule out the possibility that some other mechanisms might contribute to concurrent activation of Akt and overexpression of HER-2/neu, we compared Akt activation between the HER-2/neu-transformed NIH3T3 cell clones with that of their parental cells. In the absence of serum, Akt was activated constitutively in the HER-2/neu-transformed cells but not in the parental cells (Fig. 1 C), which confirmed that Akt was activated by the intrinsic tyrosine kinase activity ofHER-2/neu in the absence of extracellular stimulation. Furthermore, activation of Akt was blocked by wortmannin, an inhibitor of PI3K, suggesting that this HER-2/neu-mediated Akt activation occurs through PI3K. If the HER-2/neu–induced resistance to TNF is caused primarily by activation of Akt and not by other mechanisms, blocking this Akt pathway should render the cells sensitive to TNF-induced apoptosis. Therefore, to inhibit the Akt pathway, we transfected a DN-Akt (kinase-dead) DNA into the HER-2/neu-transformed NIH3T3 (HER-2/neu-3T3) cells. Upon TNF treatment, the DN-Akt transfectants of HER-2/neu-3T3 and NIH3T3 cells were about 20-fold more sensitive to apoptosis than the HER-2/neu-3T3 cells (Fig. 2 A). Expression levels of DN-Akt in these cell clones are indicated in theinsert to Fig. 2 A. To confirm the Akt anti-apoptotic effect in the HER-2/neu-overexpressed human breast cancer cells, we transfected DN-Akt DNA intoHER-2/neu-overexpressing MDA-MB453 cells and obtained several independent DN-Akt-overexpressing cell clones (Fig.2 B, insert). Similarly, the DN-Akt transfectants (clones 1 and 2) of MDA-MB453 cells became about 10-fold more sensitive to TNF-induced apoptosis than the parental cells (Fig. 2 B). Apoptosis induced by TNF was further verified by DNA fragmentation assay (Fig. 2 C). Thus, HER-2/neu was found to block TNF-induced apoptosis via the PI3K/Akt pathway. PI3K has recently been shown to be involved in the activation of transcription factor NF-κB (16.Beuaud C. Henzel W.J. Baeuerle P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 429-434Crossref PubMed Scopus (264) Google Scholar, 17.Kane L.P. Shapiro V.S. Stokoe D. Weiss A. Curr. Biol. 1999; 9: 601-604Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar), which is a p50/p65 (RelA) heterodimer regulated by its inhibitory protein, IκB (18.Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2919) Google Scholar, 19.Karin M. Delhase M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9067-9069Crossref PubMed Scopus (205) Google Scholar). Clinical evidence indicates that loss of estrogen receptor (ER) correlates strongly with overexpression of HER-2/neu (20.Tagliabue E. Menard S. Robertson J.F. Harris L. Int. J. Biol. Markers. 1999; 14: 16-26Crossref PubMed Scopus (29) Google Scholar), which is consistent with our previous finding that ER down-regulatesHER-2/neu expression (21.Russell K.S. Hung M.-C. Cancer Res. 1992; 52: 6624-6629PubMed Google Scholar). Analogously, NF-κB is often activated constitutively in ER-negative breast cancer cells (22.Nakshatri H. Bhat-Nakshatri P. Martin D.A. Goulet Jr., R.J. Sledge Jr., G.W. Mol. Cell. Biol. 1997; 17: 3629-3639Crossref PubMed Google Scholar). Thus, we hypothesized that activation of Akt by HER-2/neu may turn on NF-κB, which inhibits TNF-induced apoptosis (23.Baltimore D. Beg A.A. Science. 1996; 274: 782-784Crossref PubMed Scopus (2931) Google Scholar, 24.Wanxg C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2505) Google Scholar, 25.Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2445) Google Scholar). To test whether overexpression of HER-2/neu can activate NF-κB, we assayed the NF-κB DNA binding and transcriptional activation activities in HER-2/neu-3T3 and NIH3T3 cells and found NF-κB DNA binding activity higher in the HER-2/neu-3T3 cells than in the NIH3T3 cells, in a serum-independent manner (Fig.3 A, lanes 4 and5). As controls, NF-κB DNA binding activities were strongly activated by TNF treatment (Fig. 3 A); these activities were abrogated by the competing wild-type κB oligonucleotides (data not shown; see below). Furthermore, activation of the transcriptional activity of NF-κB in theHER-2/neu-3T3 cells without serum was confirmed by luciferase assay (Fig. 3 B). Similar results were obtained in rat HER-2/neu-transformed NIH3T3 and SW3T3 cells (data not shown). These data strongly suggested that overexpression ofHER-2/neu activates NF-κB constitutively. To determine whether sensitization of TNF-induced apoptosis in the DN-Akt transfectants occurs through inhibition of NF-κB, we measured NF-κB activities in the transfectants and MDA-MB453 cells. As shown in Fig.3, C and D, TNF-induced NF-κB DNA binding and transcription activities in the DN-Akt transfectants were significantly inhibited (3–5-fold). That these inhibitions were not caused by down-regulation of p65 or p50 by TNF is demonstrated by the finding of no change in the p65 and p50 levels of these cells in the absence or presence of TNF (Fig. 3 C, bottom panel). To investigate whether DN-Akt inhibits IκB phosphorylation and degradation, we analyzed the expression and phosphorylation patterns of IκB-α in the DN-Akt transfectants and MDA-MB453 cells before and after TNF treatment. As shown in Fig.4 A, only one IκB-α band was observed in the DN-Akt transfectants before or after the TNF treatment, whereas two bands were detected in the TNF-treated parental cells. The upper band may be the phosphorylated form of IκB-α (p-IκB-α), because it disappeared after treatment with calf intestine phosphatase (CIP, Fig. 4 B). TNF has been demonstrated to activate IκB kinases (IKKs), which in turn phosphorylate IκB, which is then degraded and activates NF-κB (26.Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (233) Google Scholar,27.Verma I.M. Stevenson J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11758-11760Crossref PubMed Scopus (179) Google Scholar). To examine whether DN-Akt blocks activation of IKKs, we compared the kinase activities of IKK-α and -β in the DN-Akt transfectants with those in the parental cells after TNF treatment, using immunocomplex kinase assays. The endogenous IKK-α and -β kinase activities were readily detected in the MDA-MB453 cells, whereas their activities were inhibited in the DN-Akt transfectants (Fig.4 C), suggesting that Akt activity is required for activation of IKKs by TNF. Furthermore, we showed that in the DN-Akt transfectants, DN-Akt and the endogenous Akt associate specifically with IKK-α in vivo regardless of TNF treatment (Fig.4 D). To further confirm that Akt is an activator upstream of IKKs, we transfected the DNA of p65 (RelA), IKK-α or -β, or a constitutively active Akt into the DN-Akt transfectants to restore TNF-induced NF-κB activities in these cells. Overexpression of each of these proteins significantly overrode the inhibitory effect of DN-Akt and restored activation of NF-κB by TNF (Fig. 4 E), indicating that Akt is indeed upstream of both IKKs. Taken together, our results suggested that Akt activity is essential for NF-κB activation by HER-2/neu and TNF. A model we propose to illustrate the parallel HER-2/neu- and TNF-induced anti-apoptotic pathways is shown in Fig. 4 F. While we were preparing this manuscript, NF-κB was reported to be a target of Akt (28.Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1883) Google Scholar, 29.Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1664) Google Scholar), confirming our finding that HER-2/neu activates the NF-κB anti-apoptotic pathway through Akt. In general, activation of the Akt signaling pathway requires extracellular survival factors (mitogenic stimuli) such as EGF, insulin, platelet-derived growth factor, thrombin, heregulin, and nerve growth factor. To our knowledge, this is the first evidence thatHER-2/neu activates the Akt/NF-κB pathway without extracellular stimulation. Our study also details a molecular mechanism of TNF resistance that may provide an interpretation for theHER-2/neu-overexpressing cancer cells, escape from host immune defenses, and the contribution of this mechanism to the poor survival of cancer patients with HER-2/neu overexpression. Understanding the HER-2/neu-mediated anti-apoptotic pathway may open an avenue for developing novel anticancer therapies forHER-2/neu-overexpressing breast and ovarian cancers.