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J Biol Chem, Vol. 275, Issue 11, 8027-8031, March 17, 2000

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

Binhua P. Zhou, Mickey C.-T. Hu, Stephanie A. Miller, Zhenming Yu, Weiya Xia, Shiaw-Yih Lin^§, and Mien-Chie Hung^¶

From the Department of Molecular and Cellular Oncology, Breast Cancer Basic Research Program, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Overexpression of HER-2/neu correlates 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 of HER-2/neu-induced TNF resistance is unknown. Here we report that HER-2/neu activates Akt and NF-B without extracellular stimulation. Blocking of the Akt pathway by a dominant-negative Akt sensitizes the HER-2/neu-overexpressing cells to TNF-induced apoptosis and inhibites IB kinases, IB phosphorylation, and NF-B activation. Our results suggested that HER-2/neu constitutively activates the Akt/NF-B anti-apoptotic cascade to confer resistance to TNF on cancer cells and reduce host defenses against neoplasia.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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 (1-5). 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 (Herceptin^TM) has been used clinically as a potent growth inhibitor of such breast cancer cells (6), and previous research has shown that overexpression of HER-2/neu up-regulates p21^Waf1 and leads to resistance by these cancer cells to Taxol (3). Still, the mechanism of HER-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), 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), pro-caspase-9 (9), and transcription factor FKHRL1 (10, 11), 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, 13).

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 in HER-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- and -, for IB phosphorylation, and for NF-B activation. Our results provide a molecular explanation for the finding that HER-2/neu-overexpressing breast cancer cells are more resistant to TNF-induced apoptosis, leading to poor prognosis and shortened survival of patients.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Lines and Cultures-- 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 human HER-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.

Apoptosis Assay-- 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, 15).

Electrophoretic Mobility Shift Assay-- Cell nuclear extracts from samples treated with or without TNF for 30 min were prepared as described previously (14, 15). 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 B 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.

Immunoprecipitation-- 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- was immunoprecipitated overnight with 2 µg of anti-IKK- 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.

Western Blot-- 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.

Immunocomplex Kinase Assay-- Cell extracts were prepared from samples treated with or without TNF, and immunocomplex kinase assays were performed as described previously (15).

Transient Transfections-- 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-B luciferase (B-luc) or mutant NF-B luciferase (mut/B-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 -galactosidase assay.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Because overexpression of HER-2/neu induced resistance to TNF (4, 5), 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 10 HER-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 10 HER-2/neu-negative tumors, 7 of 10 HER-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. 1A. 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. 1B), 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 of HER-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. 1C), which confirmed that Akt was activated by the intrinsic tyrosine kinase activity of HER-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.

View larger version (80K): [in this window] [in a new window]   Fig. 1.   HER-2/neu activates Akt. A, ten tissue sections from the HER-2/neu+ adenocarcinoma (a-d) and 10 sections from the HER-2/neu adenocarcinoma (e-h) were stained with antibodies specific to HER-2/neu (a, e), p-Akt (b, f), Akt (c, g), or normal rabbit serum (d, h) followed by immunostaining with an anti-rabbit IgG antibody conjugated with peroxidase. Antibodies were obtained from DAKO and New England Biolabs. B, nine human breast cancer cell lines were starved for 24 h without serum. Whole-cell lysates (50 µg each) were subjected to Western blot analyses using antibodies specific to HER-2/neu, Akt, p-Akt, and actin (Roche Molecular Biochemicals). Lanes 1-9, respectively: MCF-7, MCF-7/HER-2, MDA-MB435, BT483, MDA-MB231, MDA-MB453, MDA-MB361, SKBR3, and BT474. C, HER-2/neu-transformed NIH3T3 cells were established by transfecting human HER-2/neu cDNA into NIH3T3 cells. After being cultured for 3 weeks, the HER-2/neu-transformed clones (foci) were isolated and characterized by the transformed phenotypes and overexpression of HER-2/neu. Two HER-2/neu-transformed clones and parental cells were cultured in medium containing 10% fetal bovine serum or serum-free medium for 24 h, with or without wortmannin (100 nM), a PI3K inhibitor, before harvest. Whole-cell lysates were analyzed by Western blots using antibodies against Akt, p-Akt, and actin.

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. 2A). Expression levels of DN-Akt in these cell clones are indicated in the insert to Fig. 2A. To confirm the Akt anti-apoptotic effect in the HER-2/neu-overexpressed human breast cancer cells, we transfected DN-Akt DNA into HER-2/neu-overexpressing MDA-MB453 cells and obtained several independent DN-Akt-overexpressing cell clones (Fig. 2B, 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. 2B). Apoptosis induced by TNF was further verified by DNA fragmentation assay (Fig. 2C). Thus, HER-2/neu was found to block TNF-induced apoptosis via the PI3K/Akt pathway.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   HER-2/neu inhibits TNF-induced apoptosis through Akt. DN-Akt stable transfectants were generated by transfecting HER-2/neu-transformed NIH3T3 and the MDA-MB453 cells with DN-Akt cDNA (HA-tagged Akt, K179M). Several stable cell clones were isolated after G418 selection (600 µg/ml). A, NIH3T3, HER-2/neu-transformed 3T3 and its DN-Akt-transfected cell lines (clones A1 and A2) were cultured in low-serum (1% serum) medium with TNF (40 ng/ml) or without it for 48 h. Apoptotic cells were measured by flow cytometry using a fluorescence-activated cell sorter (FACS) with propidium iodide staining (mean ± S.E. in three separate experiments). Equal amounts of cell lysates were analyzed by Western blot for expression of DN-Akt in these cell clones (insert). B, MDA-MB453 cells and their DN-Akt transfectants (Clone 1 and Clone 2) were cultured in low-serum medium with (20 ng/ml) or without TNF for 48 h. Expression of DN-Akt in these clones was analyzed as described for A (insert). The apoptotic cells were quantitated by FACS (mean ± S.E. in three separate experiments) or determined by DNA fragmentation (C).

PI3K has recently been shown to be involved in the activation of transcription factor NF-B (16, 17), which is a p50/p65 (RelA) heterodimer regulated by its inhibitory protein, IB (18, 19). Clinical evidence indicates that loss of estrogen receptor (ER) correlates strongly with overexpression of HER-2/neu (20), which is consistent with our previous finding that ER down-regulates HER-2/neu expression (21). Analogously, NF-B is often activated constitutively in ER-negative breast cancer cells (22). Thus, we hypothesized that activation of Akt by HER-2/neu may turn on NF-B, which inhibits TNF-induced apoptosis (23-25). To test whether overexpression of HER-2/neu can activate NF-B, we assayed the NF-B DNA binding and transcriptional activation activities in HER-2/neu-3T3 and NIH3T3 cells and found NF-B DNA binding activity higher in the HER-2/neu-3T3 cells than in the NIH3T3 cells, in a serum-independent manner (Fig. 3A, lanes 4 and 5). As controls, NF-B DNA binding activities were strongly activated by TNF treatment (Fig. 3A); these activities were abrogated by the competing wild-type B oligonucleotides (data not shown; see below). Furthermore, activation of the transcriptional activity of NF-B in the HER-2/neu-3T3 cells without serum was confirmed by luciferase assay (Fig. 3B). Similar results were obtained in rat HER-2/neu-transformed NIH3T3 and SW3T3 cells (data not shown). These data strongly suggested that overexpression of HER-2/neu activates NF-B constitutively. To determine whether sensitization of TNF-induced apoptosis in the DN-Akt transfectants occurs through inhibition of NF-B, we measured NF-B activities in the transfectants and MDA-MB453 cells. As shown in Fig. 3, C and D, TNF-induced NF-B 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. 3C, bottom panel).

View larger version (44K): [in this window] [in a new window]   Fig. 3.   HER-2/neu activates NF-B. A, NIH3T3 cells and HER-2/neu-transformed 3T3 cells were cultured in serum-containing or serum-free medium for 24 h. Nuclear extracts (5 µg each) were used to determine NF-B DNA binding activities by an electrophoretic mobility shift assay using an oligonucleotide probe containing the B binding site. Nuclear extracts from the TNF-stimulated cells were included as positive controls. B, NIH3T3 and HER-2/neu-transformed 3T3 cells were cotransfected with 0.2 µg of pcDNA3-lacZ plus 1.8 µg of either wild-type or mutant NF-B luciferase (B-luc or mut/B-luc) plasmids. Forty-eight h post-transfection, luciferase activities were determined and normalized by -galactosidase activities. NF-B activities were calculated by the luciferase activities of B-luc versus mut/B-luc (mean ± S.E. in three separate experiments). C, DN-Akt blocks activation of NF-B induced by TNF. The MDA-MB453 cells and DN-Akt transfectants (Clone 1 and Clone 2) were treated with (20 ng/ml) or without TNF for 5 min, and NF-B DNA binding activities were determined as described above for panel A. Cold wild-type or mutant NF-B oligonucleotides were included as controls in the TNF-induced MDA-MB453 cells. An anti-p65 antibody (Santa Cruz) was also included in the assay; the supershifted complex is indicated by an arrow. As control, 50 µg of each cell lysate was assayed for the expression of p65 or p50 by Western blots using anti-p65 or anti-p50 antibody (bottom panel). D, MDA-MB453 cells and DN-Akt transfectants were cotransfected with pcDNA3-lacZ plus B-luc or mut/B-luc plasmids as described above. After 40 h of transfection, the cells were treated with or without TNF (20 ng/ml) for 8 h and then harvested. NF-B activities were determined as above.

To investigate whether DN-Akt inhibits IB phosphorylation and degradation, we analyzed the expression and phosphorylation patterns of IB- in the DN-Akt transfectants and MDA-MB453 cells before and after TNF treatment. As shown in Fig. 4A, only one IB- 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 IB- (p-IB-), because it disappeared after treatment with calf intestine phosphatase (CIP, Fig. 4B). TNF has been demonstrated to activate IB kinases (IKKs), which in turn phosphorylate IB, which is then degraded and activates NF-B (26, 27). To examine whether DN-Akt blocks activation of IKKs, we compared the kinase activities of IKK- and - in the DN-Akt transfectants with those in the parental cells after TNF treatment, using immunocomplex kinase assays. The endogenous IKK- and - kinase activities were readily detected in the MDA-MB453 cells, whereas their activities were inhibited in the DN-Akt transfectants (Fig. 4C), 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- in vivo regardless of TNF treatment (Fig. 4D). To further confirm that Akt is an activator upstream of IKKs, we transfected the DNA of p65 (RelA), IKK- or -, or a constitutively active Akt into the DN-Akt transfectants to restore TNF-induced NF-B activities in these cells. Overexpression of each of these proteins significantly overrode the inhibitory effect of DN-Akt and restored activation of NF-B by TNF (Fig. 4E), indicating that Akt is indeed upstream of both IKKs. Taken together, our results suggested that Akt activity is essential for NF-B 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. 4F. While we were preparing this manuscript, NF-B was reported to be a target of Akt (28, 29), confirming our finding that HER-2/neu activates the NF-B anti-apoptotic pathway through Akt.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   HER-2/neu activates the Akt/NF-B signaling cascade. A, MDA-MB453 and DN-Akt transfectants were starved for 12 h without serum and treated with TNF (20 ng/ml) or without it for 5 min before harvesting. Equal amounts of whole-cell lysates were subjected to SDS-PAGE (12%) and transferred to nitrocellulose membranes. IB- was detected by Western blot using an anti-IB- antibody (Santa Cruz). B, phosphorylation of IB- induced by TNF could be dephosphorylated by a phosphatase. Cell lysates (500 µg each) from the TNF-treated MDA-MB453 cells were immunoprecipitated with anti-IB-, and the precipitates were then incubated with or without 20 units of calf intestine phosphatase (CIP) at 37 °C for 30 min before Western blot analysis with anti-IB-. C, MDA-MB453 and DN-Akt transfectants were starved and treated with TNF (10 min). Endogenous IKK- and - were immunoprecipitated with anti-IKK- and anti-IKK- antibodies, respectively, and their kinase activities were determined by immunocomplex kinase assays using GST-IB- (amino acid 1-54) as a substrate as described previously (15). D, MDA-MB453 and DN-Akt transfectants (Clone 1) were treated with TNF (20 ng/ml) for different time periods. The endogenous IKK- was immunoprecipitated with an anti-IKK- antibody, then analyzed by Western blot (WB) using an anti-Akt, anti-HA, or anti-IKK- antibody. E, NF-B activity in the DN-Akt transfectants could be partially restored by overexpression of p65, IKK-, IKK-, or a constitutively active Akt (CA-Akt). The DN-Akt transfectants (clone 1) were cotransfected with pcDNA3-lacZ (0.2 µg) and either B-luc or mut/B-luc (0.6 µg each) plus p65, IKK-, IKK-, or CA-Akt plasmid (1.2 µg each). Forty h post-transfection, the cells were treated with or without TNF, and the NF-B activities were determined as described above for panel B in Fig. 3 (mean ± S.E. in four separate experiments). F, model of HER-2/neu activation of the Akt/NF-B pathway that blocks TNF-induced apoptosis. TNFR, TNF receptor; NIK, NF-B-inducing kinase.

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 that HER-2/neu activates the Akt/NF-B pathway without extracellular stimulation. Our study also details a molecular mechanism of TNF resistance that may provide an interpretation for the HER-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 for HER-2/neu-overexpressing breast and ovarian cancers.

	FOOTNOTES

^* This work was supported by Grants R01-CA58880 and R01-CA77858 (to M.-C. H.) and Cancer Core Grant 16672 from the NCI, National Institutes of Health, by the Nellie Connally Breast Cancer Research Fund, and by a Faculty Achievement Award at M. D. Anderson Cancer Center (to M.-C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These two authors are recipients, respectively, of postdoctoral and predoctoral fellowships from the United States Department of Defense (DOD) Breast Cancer Research Training Grant (DAMD17-99-1-9264).

^§ Predoctoral fellow of the DOD Breast Cancer Research Program (DAMD17-98-1-8242).

^¶ To whom correspondence should be addressed. Tel.: 713-792-3668; Fax: 713-794-0209; E-mail: mhung@notes.mdacc.tmc.edu.

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; EGF, epidermal growth factor; IKK, IB kinase; HA, hemagglutinin; luc, luciferase; PBS, phosphate-buffered saline; DN-Akt, dominant-negative Akt; p-Akt, phosphorylated Akt; ER, estrogen receptor; NF-B, nuclear factor-B; PAGE, polyacrylamide gel electrophoresis; PI3K, phosphatidylinositol 3-kinase; 3T3 cells, NIH3T3 cells.

	REFERENCES

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				N. E. Martin, T. B. Brunner, K. D. Kiel, T. F. DeLaney, W. F. Regine, M. Mohiuddin, E. F. Rosato, D. G. Haller, J. P. Stevenson, D. Smith, et al. A Phase I Trial of the Dual Farnesyltransferase and Geranylgeranyltransferase Inhibitor L-778,123 and Radiotherapy for Locally Advanced Pancreatic Cancer Clin. Cancer Res., August 15, 2004; 10(16): 5447 - 5454. [Abstract] [Full Text] [PDF]

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