NF-κB inhibition markedly enhances sensitivity of resistant breast cancer tumor cells to tamoxifen

      Abstract

      Studies show that high Akt activity in breast carcinoma is associated with endocrine therapy resistance. Breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions, and are resistant to the growth inhibitory effects of tamoxifen. Understanding the targets of Akt signaling mediating tamoxifen resistance is of clinical significance. One possible target is nuclear factor kappa B(NF-κB), a transcription factor that plays a critical role in resistance to apoptosis and the induction of angiogenesis and invasion. In the present study, we found that Akt activity correlated with phosphorylation of IκB (the negative regulator of NF-κB), NF-κB DNA binding and tamoxifen resistance in vivo. Importantly, we found that co-treatment with the NF-κB inhibitor, parthenolide, or overexpression of IκB superrepressor restored tamoxifen sensitivity to our refractory Akt MCF-7 cells. These data suggest that activation of NF-κB via the PI3K/Akt signaling pathway may be a significant mechanism for development of endocrine therapy resistance in breast cancer, and that inhibition of NF-κB may be an effective treatment strategy to limit the progression of this disease.

      Keywords

      Introduction

      The PKB/Akt kinase pathway is activated in cells exposed to diverse stimuli such as hormones, growth factors and extracellular matrix components [
      • Tsai E.M.
      • Wang S.C.
      • Lee J.N.
      • Hung M.C.
      Akt activation by estrogen in estrogen receptor-negative breast cancer cells.
      ,
      • Gu J.
      • Fujibayashi A.
      • Yamada K.M.
      • Sekiguchi K.
      Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways.
      ]. The PKB family of serine/threonine protein kinases have been implicated in mediating a variety of biological responses that are central to the process of oncogenic transformation of mammalian cells, including inhibiting apoptosis and stimulating cellular growth (reviewed in [
      • Nicholson K.M.
      • Anderson N.G.
      The protein kinase B/Akt signalling pathway in human malignancy.
      ]). Aberrant activation of the PKB/Akt pathway can suppress the apoptotic response, undermine cell cycle control and selectively enhance the production of key growth and survival factors [
      • Hutchinson J.
      • Jin J.
      • Cardiff R.D.
      • et al.
      Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression.
      ]. It has been shown by our group, as well as by others, that PKB/Akt can protect breast cancer cells from tamoxifen-induced apoptosis [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ,
      • Clark A.S.
      • West K.
      • Streicher S.
      • Dennis P.A.
      Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells.
      ]. Multiple laboratories have now demonstrated that the PI3K/Akt pathway provides these cell survival signals, in part, through activation of the nuclear factor kappa B (NF-κB) transcription factor [
      • Madrid L.V.
      • Mayo M.W.
      • Reuther J.Y.
      • Baldwin Jr, A.S.
      Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-κB through utilization of the IκB kinase and activation of the mitogen activated protein kinase p38.
      ].
      In previous studies we demonstrated that expression of a constitutively active Akt [
      • Kohn A.D.
      • Takeuchi F.
      • Roth R.A.
      Akt, a pleckstrin homology domain containing kinase.
      ] confers hormone-independence to MCF-7 breast cancer cells (myrAkt1 MCF-7) [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ]. These cells growin vitro in charcoal-stripped serum, and are able to grow in vivo as xenografts without estrogen supplementation. In addition, these cells are resistant in vitro, as well as in vivo, to the inhibitory effects of the antiestrogen, tamoxifen [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ], by mechanisms that we are currently investigating. As part of these studies, we examined differences in expression and/or activity of downstream targets of Akt signaling, including the NF-κB transcription factor. We found that the tamoxifen-resistant, constitutively active Akt MCF-7 cells demonstrated significantly higher levels of phosphorylated IκB, as well as higher levels of NF-κB DNA binding and transcriptional activity, compared with levels observed in the control MCF-7 cells. Inhibition of NF-κB activity, either pharmacologically or molecularly, restored tamoxifen sensitivity to the resistant cell line. These data suggest that activation of NF-κB via the PI3K/Akt signaling pathway may be a significant mechanism for development of endocrine therapy resistance in breast cancer, and that inhibition of NF-κB may be an effective treatment strategy for this relatively resistant disease.

      Materials and methods

       Cell lines

      The control and myrAkt1 MCF-7 breast cancer cells, stably transfected with an empty pCDAN3.1 (+) vector (Invitrogen, Carlsbad, CA, USA) or the myrAkt Δ4-129 (a kind gift from Richard A. Roth, Stanford University School of Medicine, Stanford, CA, USA) [
      • Kohn A.D.
      • Takeuchi F.
      • Roth R.A.
      Akt, a pleckstrin homology domain containing kinase.
      ], respectively, have been described previously [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ]. Stable transfectant cell lines were maintained in improved minimal essential medium (Invitrogen) supplemented with 5% complete fetal bovine serum (FBS) (Sigma, St Louis, MO, USA), 5% charcoal-stripped FBS (Hyclone, Logan, UT, USA), 6 ng/ml of bovine insulin (Sigma) and 400 mg/l G418.

       Western blot analysis

      Western blot analyses were carried out as described previously [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ]. Protein lysates were subjected to immunodetection with antibodies to phosphorylated and then total Akt (Cell Signaling Technology, Beverly, MA, USA), phosphorylated, and then total IκB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and finally actin (Santa Cruz Biotechnology) for a loading control. Signal detection was carried out using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA).

       Electrophoretic mobility shift assay

      NF-κB DNA binding activity was measured in the nuclear protein extracts by electrophoretic mobility shift assay (EMSA), as described earlier [
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • et al.
      Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.
      ]. A double-stranded oligonucleotide (NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′; Santa Cruz Biotechnology) containing the decameric consensus sequence,5′-GGGACTTTCC-3′, was used as a probe. For the competition assay, the protein extract (10 µg) was preincubated with homologous unlabeled oligonucleotide for 5 min on ice and the labeled probe was then added. Absence of protein extract, competition with 100-fold molar excess unlabeled NF-κB, and mutant NF-κB oligo (5′-AGTTGAGGCGACTTTCCCAGGC-3′; Santa Cruz Biotechnology) served as controls. Protein extracts were from cultured cells, control MCF-7 xenografts from tamoxifen-treated mice, cultured cells transiently transfected with 10 µg of either an empty expression vector or an expression plasmid for the non-degradable IκB (S32A,S36A; a kind gift from Inder M. Verma, The Salk Institute for Biological Studies, San Diego, CA, USA) [
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • et al.
      Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.
      ] or cells treated for 24 h with 2 µM parthenolide (Santa Cruz Biotechnology).

       Luciferase reporter assay

      Transient transfections were performed three or more times in triplicate wells. Cells were seeded in six-well cluster plates (Falcon, Franklin Lakes, NJ, USA) at a density of 2 × 105 cells/well 24 h prior to transfection. Three microliters of FuGene 6 transfection reagent (Boeringher Mannheim, Indianapolis, IN, USA) were used to transfect 1 µg of the 5× NF-κB luciferase reporter plasmid (Stratagene, La Jolla, CA, USA). The plasmid pNull-Renilla (Promega, Madison, WI, USA), an expression plasmid for the Renilla luciferase gene void of eukaryotic promoter or enhancer sequences, was co-transfected for transfection normalization. One microgram of HA-Akt K179M expression plasmid was co-transfected with the 5× NF-κB luciferase reporter where indicated. Total concentration of transfected DNA was corrected by addition of pCDNA3.1 vector (Invitrogen). Cells were allowed to recover for 24 h after transfection, then transferred to either serum-free or 10% FBS-containing media. Luciferase activity was assessed 24 h later. All transfections are reported as fold activity over control after normalization for Renilla expression. Control is the basal activity of the 5× NF-κB luciferase reporter in the control cells under serum-starved conditions. Luciferase activity was measured using the Dual Luciferase kit (Promega), as per the manufacturer’s instructions. A combination of three independent experiments is shown.

       Growth proliferation assay

      Cell growth was assessed by MTT [3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide] (Sigma) dye conversion, as described previously [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ]. For molecular inhibition studies, cells were transiently transfected using FuGene 6 with increasing concentrations of the IκB (S32A, S36A) expression plasmid alone, treated with 10–7 M tamoxifen alone, or transiently transfected with increasing concentrations of the IκB (S32A, S36A) expression plasmid in the presence of 10–7 M tamoxifen. Total concentration of transfected DNA was corrected by addition of pCDNA3.1 vector. For pharmacological studies, cells were treated with increasing concentrations of tamoxifen alone, or in combination with 2 µM parthenolide. All studies were carried out in the presence of 10% (half complete, half charcoal-stripped) FBS supplemented with 6 ng/ml insulin. Growth was assessed after 96 h of continuous treatment. Data are presented as: 100 – (percentage growth compared with untreated and/or vector transfected control), and are the combination of three independent experiments.

       Statistical analysis

      For luciferase and MTT analyses, Student’s t-test was used.

      Results

       High Akt activity correlates with increased IκBα phosphorylation, NF-κB DNA binding and tamoxifen resistance

      We have previously demonstrated that the myrAkt1 MCF-7 cell line model was tamoxifen-resistant [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
      ]. To determine the mechanisms by which Akt induces resistance, we investigated the downstream targets of Akt signaling, including NF-κB. Western blot analysis (Figure 1A) of lysates from control and myrAkt1 MCF-7 cells demonstrated significantly higher levels of phosphorylated IκBα in the myrAkt1 MCF-1 cell lines compared with that observed in the control cells, correlating with the high levels of phosphorylated myrAkt1 expressed in these cells. The myrAkt1 protein does not contain the PH domain, and therefore runs faster than the wild-type Akt1 on SDS-polyacrylamide gels. EMSAs (Figure 1B) performed on lysates from control (lane D) and myrAkt1 (lanes E and F) MCF-7 cells showed higher levels of NF-κB DNA binding in the myrAkt1 cell lines (60% and 57%, respectively) compared with that observed in the control cells. EMSA analysis of lysates from xenograft MCF-7 breast cancer tumors from mice treated 17 days with 500 µg/day tamoxifen [
      • deGraffenried L.A.
      • Friedrichs W.E.
      • Fulcher L.
      • et al.
      Molecular Targets and Cancer Therapeutics.
      ] (lanes G–J) demonstrated lower levels of NF-κB DNA binding in the tumors that responded to tamoxifen (lanes G–I) (31%, 27% and 23%, respectively) compared with the higher level of NF-κB DNA binding demonstrated in the tamoxifen-resistant tumor(lane J). These data indicate an association between Akt activity, NF-κB DNA binding and tamoxifen resistance.
      Figure 1
      Figure 1Akt status correlates with nuclear factor kappa B (NF-κB) DNA binding and tamoxifen response. (A) Western blot analyses of lysates from control and myrAkt1 MCF-7 cells probed with antibodies to phospho-473 Akt, total Akt, phospho-IκBα, total IκBα and actin. (B) Electrophoretic mobility shift assays were performed using control (lanes A, B and D) and myrAkt1 (lanes E and F) MCF-7 cell extracts, or extracts from MCF-7 orthotopic tumors from mice treated with tamoxifen (lanes G–J). Densitometry units are presented relative to levels obtained from the control lysates in lane D. A double-stranded oligonucleotide containing the decameric NF-κB consensus sequence was used as a probe and was incubated with 10 µg of extract alone (lanes D–J), or in the presence of 100-fold molar excess of a mutant NF-κB oligonucleotide (lane A) or cold oligonucleotide (lane B). Lane C contains no protein.

       NF-κB transcriptional activity in MCF-7 breast cancer cells is regulated by Akt

      In order to determine whether the higher level of NF-κB DNA binding was correlated with an increase in transcriptional activity of NF-κB, we transiently transfected the 5× NF-κB–luciferase reporter plasmid into control and myrAkt1 MCF-7 cells (Figure 2). We found that the myrAkt1 (dark gray bars) cells demonstrated a >50% higher level of NF-κB activity compared with that in the control cells (white bars) under both serum-starved and stimulated (10% FBS) conditions. Expression of a kinase-defective mutant of Akt, HA-Akt K179M [
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • et al.
      The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.
      ], decreased NF-κB by more than 85% in both cell lines, indicating a direct relationship between Akt activity and transcriptional activity of NF-κB.
      Figure 2
      Figure 2Nuclear factor kappa B (NF-κB) transcriptional activity is regulated by Akt. A luciferase reporter construct containing 5× direct repeats of a binding site for NF-κB was transiently transfected into control (white bars) or myrAkt1 (gray bars) MCF-7 cells. One set of cells (K179M) was co-transfected with the kinase-mutant K179M Akt1. Transfected cells were grown for 24 h under conditions of either serum starvation (serum starved) or in the presence of 10% fetal bovine serum (10% FBS and K179M). NF-κB promoter activity was normalized to Renilla activity and is expressed as relative fold luciferase units over control, and is the combination of three independent experiments that were carried out in triplicate. All values had a statistical difference P ≤0.05

       Interference with IκB degradation decreases NF-κB DNA binding

      Since Akt signaling induces phosphorylation of IκB, resulting in its ubiquitination and subsequent degradation by the 26S proteasome [
      • Zandi E.
      • Karin M.
      Bridging the gap: composition, regulation, and physiological function of the Ikappa B kinase complex.
      ], freeing NF-κB to translocate to the nucleus, we assessed the effects of blocking IκB degradation on NF-κB DNA binding (Figure 3). EMSA analysis of lysates from control (lanes D, F, H and J) and myrAkt1 (lanes E, G, I and K) MCF-7 cells demonstrated that NF-κB DNA binding was much lower in the control MCF-7 cells (lane D) compared with that in the myrAkt1 cells (lane E). Treatment with 10–7 M tamoxifen (lanes F and G) appeared to slightly increase NF-κB DNA binding in the control MCF-7 cell line. The mechanism and significance for this observation is as yet unclear, but others have observed that tamoxifen treatment of estrogen receptor (ER)α-positive breast cancer cells results in short-term activation of Akt [
      • Clark A.S.
      • West K.
      • Streicher S.
      • Dennis P.A.
      Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells.
      ]. Transient transfection of a non-phosphorable IκBα (S32A/S36A) [
      • Chandrasekar B.
      • Melby P.C.
      • Sarau H.M.
      • et al.
      Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.
      ] (lanes H and I) or treatment with 2 µM of the proteasome inhibitor, parthenolide (lanes J and K), reduced the very high levels of NF-κB DNA binding observed in the myrAkt1 cells by 33% and 50%, respectively, to levels similar to those observed in the control cells.
      Figure 3
      Figure 3Nuclear factor kappa B (NF-κB) DNA binding is inhibited by blockers of IκB degradation. Electrophoretic mobility shift assays were performed using lysates from control (lanes D, F, H and J) and myrAkt1 (lanes A, B, E, G, I and K) MCF-7 cells probed with a double-stranded oligonucleotide containing the decameric NF-κB consensus sequence. Cells were transfected with either an empty vector (lanes D and E), or an expression plasmid for the non-phosphorable IκB (S32,36A) (lanes H and I) treated with 10–7 M tamoxifen (lanes F and G) or with 2 µM parthenolide (lanes J and K). Lanes A and B are controls of 100-fold competition with either a mutant NF-κB probe or a cold NF-κB probe, respectively. Densitometry units are presented relative to values obtained with control lysate (lane D).

       Molecular and pharmacological inhibition of NF-κB restores tamoxifen response in cells with high Akt activity

      In order to assess how inhibition of NF-κB DNA binding would effect tamoxifen response, we performed a series of MTT proliferation assays using both molecular (Figure 4A) and pharmacological (Figure 4B) mechanisms to block NF-κB activity. As seen in Figure 4A, transient expression of increasing concentrations (1–20 µg/ml) of the non-degradable IκB (S32A/S36A) resulted in a concentration-dependent increase in growth inhibition of control (white bars) and myrAkt1 cell lines 12 and 13 (light and dark gray bars, respectively). Tamoxifen at a concentration of 10–7 M was not significantly growth inhibitory to either of the myrAkt1 MCF-7 cell lines. When tamoxifen treatment was combined with expression of IκB (S32A/S36A), the myrAkt1 cells demonstrated a growth inhibitory response that was greater than that observed at the same concentration of DNA without treatment. In Figure 4B, the myrAkt1 MCF-7 cells showed no growth inhibitory response to increasing concentrations of tamoxifen (10–9–10–7 M), as measured by MTT analysis. Treatment with 2 µM parthenolide inhibited growth in the control cells by almost 30%, and the myrAkt1 cell lines by ∼10%. However, when the parthenolide was combined with tamoxifen, the myrAkt1 cells demonstrated a significant and dose-dependent growth inhibitory response to increasing concentrations of tamoxifen. These data suggest that interference with NF-κB transcriptional activity could restore tamoxifen response in tumors with high Akt activity.
      Figure 4
      Figure 4Inhibition of nuclear factor kappa B (NF-κB) restores tamoxifen response in cells with high Akt activity. Growth inhibition of control (white bars) and myrAkt1 clones 12 and 13 (light gray and dark gray bars, respectively) MCF-7 cells was measured by MTT [3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide] analysis and is presented as: 100 – (relative percentage of growth compared with vector transfection and no drug treatment after). (A) Transfection with increasing concentrations (1–20 µg/ml) of the non-phosphorable IκB (S32,36A) either alone or in the presence of 10–7 M tamoxifen; (B) treatment with increasing concentrations of tamoxifen (10–9–10–6 M) either alone or in the presence of 2 µM parthenolide. Presented is the combination of three independent experiments that were carried out in triplicate. *P ≤0.05 compared with control value.

      Discussion

      In the present study, we have observed a strong correlation between tamoxifen resistance, Akt kinase activity and NF-κB activity, adding to the growing body of evidence indicating an important role for Akt in the development of tamoxifen resistance and proposing a potentially novel, non-ERα-mediated mechanism by which Akt confers resistance. Previous breast cancer studies have proposed a potential role for NF-κB in the progression of hormone-dependent cancers to hormone independence. Constitutive activation of NF-κB was found in ERα-negative breast cancer cell lines and poorly differentiated primary tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, non-malignant phenotype to an ER-negative, malignant phenotype was accompanied by constitutive activation of NF-κB [
      • Nakshatri H.
      • Bhat-Nakshatri P.
      • Martin D.
      • et al.
      Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.
      ].
      NF-κB activation has been connected with multiple aspects of oncogenesis, including the control of apoptosis, the cell cycle, differentiation and cell migration (reviewed in [
      • Garg A.
      • Aggarwal B.B.
      Nuclear transcription factor-kappaB as a target for cancer drug development.
      ]). NF-κB is a transcription factor the activity of which is tightly regulated at multiple levels [
      • Zandi E.
      • Karin M.
      Bridging the gap: composition, regulation, and physiological function of the Ikappa B kinase complex.
      ,
      • Baldwin Jr, A.S.
      The NF-kappa B and I kappa B proteins: new discoveries and insights.
      ]. In most cell types, NF-κB complexes are normally sequestered in the cytoplasm as an inactive complex bound by an inhibitor known as IκB, until a cell is activated by a relevant stimulus [
      • Baldwin Jr, A.S.
      The NF-kappa B and I kappa B proteins: new discoveries and insights.
      ]. Following cellular stimulation, IκB proteins become phosphorylated by the IκB kinase (IKK). This phosphorylation of IκB results in its ubiquitination and subsequent degradation by the 26S proteasome [
      • Zandi E.
      • Karin M.
      Bridging the gap: composition, regulation, and physiological function of the Ikappa B kinase complex.
      ]. The degradation of IκB proteins liberates NF-κB, allowing its translocation to the nucleus, blocking cell-death pathways (reviewed in [
      • Baldwin A.S.
      Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB.
      ]). Recent studies have now suggested the NF-κB activation is an important mechanism for chemotherapy resistance, and that inhibition of NF-κB significantly enhances tumor cell response to chemotherapeutic agents [
      • Ma M.H.
      • Yang H.H.
      • Parker K.
      • et al.
      The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents.
      ].
      Activation of the Akt pathway has been reported to stimulate IKK-dependent IκB degradation and nuclear translocation of NF-κB [
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • et al.
      NF-kappaB activation by tumour necrosis factor requires the Akt serine–threonine kinase.
      ]. In our studies, we observed a correlation between Akt activity and the phosphorylation of IκBα, suggesting that in our model, regulation of NF-κB is at least in part through Akt regulation of IκB degradation. Studies are ongoing to delineate the exact mechanisms by which Akt modulates NF-κB activity. However, although the mechanism(s) remains to be determined, it is clear from our studies, as well as those done in other laboratories [
      • Bhat-Nakshatri P.
      • Sweeney C.J.
      • Nakshatri H.
      Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells.
      ], that Akt activation of NF-κB leads to invasive and drug-resistant growth of breast cancer, suggesting that targeting this pathway may be extremely effective as a therapeutic approach for the treatment of hormone refractory breast cancer.
      Several newly developed proteasome inhibitors suppress NF-κB activity by inhibiting IκBα degradation, correlating with antitumor activity against both solid and hematologic tumor types [
      • Adams J.
      • Palombella V.J.
      • Elliott P.J.
      Proteasome inhibition: a new strategy in cancer treatment.
      ]. Importantly, in phase I and II clinical trials for patients with chemoresistant multiple myeloma, the proteosome inhibitor PS-341 (Millennium Inc., Boston, MA, USA) showed antitumor responses [
      • Singhal S.
      • Mehta J.
      • Desikan R.
      • et al.
      Antitumor activity of thalidomide in refractory multiple myeloma.
      ,
      • Orlowski R.Z.
      • Stinchcombe T.E.
      • Mitchell B.S.
      • et al.
      Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.
      ], and has recently received approval from the US Food and Drug Administration for the treatment of multiple myeloma patients who have received at least two prior therapies and who have demonstrated disease progression on the last therapy. In addition, there are currently several phase II clinical trials ongoing to evaluate the potential of NF-κB inhibitors to enhance or restore therapeutic response to a wide range of therapeutics, including platinum drugs, mitoxantrone and doxorubicin.
      Our studies indicate that Akt activation of NF-κB may be an important mechanism in the development of tamoxifen-resistant breast cancer. Several studies have also now demonstrated that NF-κB plays a role in blocking the efficacy of cancer chemotherapies and radiation [
      • Baldwin A.S.
      Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB.
      ]. Activation of NF-κB is emerging as one of the major mechanisms of tumor cell resistance to cytokines and chemotherapeutic agents. Our data suggest that it is also a major mechanism of tumor cell resistance to antihormones. Recent studies using combinations with other agents suggest that combination regimens can have great efficacy [
      • Figg W.D.
      • Arlen P.
      • Gulley J.
      • et al.
      A randomized phase II trial of docetaxel (taxotere) plus thalidomide in androgen-independent prostate cancer.
      ]. Our data, in which we found that the use of NF-κB inhibitors restores tamoxifen response in the tamoxifen-resistant cells, indicate that a combination regimen of tamoxifen with NF-κB inhibitors may have great efficacy in a subset of resistant breast cancers.
      However, there are concerns regarding the targeting of NF-κB for cancer treatment. These concerns range from the fact that current NF-κB inhibitors are not specific to issues regarding the potential proapoptotic and critical immunological functions of NF-κB. Moreover, the role of NF-κB could be different in different tumor types. Although rare, there are systems in which NF-κB has been shown to play a proapoptotic role in addition to its more common antiapoptotic role [
      • Dumont A.
      • Hehner S.P.
      • Hofmann T.G.
      • et al.
      Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB.
      ]. These concerns strongly indicate that further studies are necessary to dissect the roles of NF-κB in a variety of cancers, and to determine the applicability of inhibiting NF-κB as an adjuvant approach in standard approaches to cancer therapy. For this reason, further investigation is necessary into the role of NF-κB activation through the Akt pathway in the development of hormone refractory breast cancer, and the potential use of agents that target NF-κB as an effective treatment strategy for this relatively resistant disease.

      Acknowledgements

      This work was supported in part by grants to L.A.d. by the Susan G. Komen Foundation, PDF 2000 655, the San Antonio Area Foundation and the Shelby Rae Tengg Foundation, and to B.C. by American Heart Association Grant-in-Aid 0150105N and National Institutes of Health Grant HL68020.

      references

        • Tsai E.M.
        • Wang S.C.
        • Lee J.N.
        • Hung M.C.
        Akt activation by estrogen in estrogen receptor-negative breast cancer cells.
        Cancer Res. 2001; 61: 8390-8392
        • Gu J.
        • Fujibayashi A.
        • Yamada K.M.
        • Sekiguchi K.
        Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways.
        J Biol Chem. 2002; 277: 19922-19928
        • Nicholson K.M.
        • Anderson N.G.
        The protein kinase B/Akt signalling pathway in human malignancy.
        Cell Signal. 2002; 14: 381-395
        • Hutchinson J.
        • Jin J.
        • Cardiff R.D.
        • et al.
        Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression.
        Mol Cell Biol. 2001; 21: 2203-2212
        • deGraffenried L.A.
        • Friedrichs W.E.
        • Fulcher L.
        • et al.
        Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.
        Ann Oncol. 2003; 14: 1051-1056
        • Clark A.S.
        • West K.
        • Streicher S.
        • Dennis P.A.
        Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells.
        Mol Cancer Ther. 2002; 1: 707-717
        • Madrid L.V.
        • Mayo M.W.
        • Reuther J.Y.
        • Baldwin Jr, A.S.
        Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-κB through utilization of the IκB kinase and activation of the mitogen activated protein kinase p38.
        J Biol Chem. 2001; 20: 20
        • Kohn A.D.
        • Takeuchi F.
        • Roth R.A.
        Akt, a pleckstrin homology domain containing kinase.
        is activated primarily by phosphorylation. J Biol Chem. 1996; 271: 21920-21926
        • Chandrasekar B.
        • Melby P.C.
        • Sarau H.M.
        • et al.
        Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.
        J Biol Chem. 2003; 278: 4675-4686
        • deGraffenried L.A.
        • Friedrichs W.E.
        • Fulcher L.
        • et al.
        Molecular Targets and Cancer Therapeutics.
        The mTOR inhibitor, CCI-779, restores tamoxifen response in breast cancer cells with high Akt activity. 2002 (Frankfurt, Germany)
        • Franke T.F.
        • Yang S.I.
        • Chan T.O.
        • et al.
        The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.
        Cell. 1995; 81: 727-736
        • Zandi E.
        • Karin M.
        Bridging the gap: composition, regulation, and physiological function of the Ikappa B kinase complex.
        Mol Cell Biol. 1999; 19: 4547-4551
        • Nakshatri H.
        • Bhat-Nakshatri P.
        • Martin D.
        • et al.
        Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.
        Mol Cell Biol. 1997; 17: 3629-3639
        • Garg A.
        • Aggarwal B.B.
        Nuclear transcription factor-kappaB as a target for cancer drug development.
        Leukemia. 2002; 16: 1053-1068
        • Baldwin Jr, A.S.
        The NF-kappa B and I kappa B proteins: new discoveries and insights.
        Annu Rev Immunol. 1996; 14: 649-683
        • Baldwin A.S.
        Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB.
        J Clin Invest. 2001; 107: 241-246
        • Ma M.H.
        • Yang H.H.
        • Parker K.
        • et al.
        The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents.
        Clin Cancer Res. 2003; 9: 1136-1144
        • Ozes O.N.
        • Mayo L.D.
        • Gustin J.A.
        • et al.
        NF-kappaB activation by tumour necrosis factor requires the Akt serine–threonine kinase.
        Nature. 1999; 401: 82-85
        • Bhat-Nakshatri P.
        • Sweeney C.J.
        • Nakshatri H.
        Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells.
        Oncogene. 2002; 21: 2066-2078
        • Adams J.
        • Palombella V.J.
        • Elliott P.J.
        Proteasome inhibition: a new strategy in cancer treatment.
        Invest New Drugs. 2000; 18: 109-121
        • Singhal S.
        • Mehta J.
        • Desikan R.
        • et al.
        Antitumor activity of thalidomide in refractory multiple myeloma.
        N Engl J Med. 1999; 341: 1565-1571
        • Orlowski R.Z.
        • Stinchcombe T.E.
        • Mitchell B.S.
        • et al.
        Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.
        J Clin Oncol. 2002; 20: 4420-4427
        • Figg W.D.
        • Arlen P.
        • Gulley J.
        • et al.
        A randomized phase II trial of docetaxel (taxotere) plus thalidomide in androgen-independent prostate cancer.
        Semin Oncol. 2001; 28: 62-66
        • Dumont A.
        • Hehner S.P.
        • Hofmann T.G.
        • et al.
        Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB.
        Oncogene. 1999; 18: 747-757