1 Department of Medicine, UT Health Science Center at San Antonio, San Antonio, TX; 2 The Johns Hopkins Oncology Center, Johns Hopkins Medical Center, Baltimore, MD, USA
Received 7 January 2004; revised 17 February 2004; accepted 18 February 2004
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ABSTRACT |
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Key words:
Akt, breast cancer, NF-B, tamoxifen resistance
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Introduction |
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In previous studies we demonstrated that expression of a constitutively active Akt [8] confers hormone-independence to MCF-7 breast cancer cells (myrAkt1 MCF-7) [5]. These cells grow in 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 [5], 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-B transcription factor. We found that the tamoxifen-resistant, constitutively active Akt MCF-7 cells demonstrated significantly higher levels of phosphorylated I
B, as well as higher levels of NF-
B DNA binding and transcriptional activity, compared with levels observed in the control MCF-7 cells. Inhibition of NF-
B activity, either pharmacologically or molecularly, restored tamoxifen sensitivity to the resistant cell line. These data suggest that activation of NF-
B 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-
B may be an effective treatment strategy for this relatively resistant disease.
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Materials and methods |
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Western blot analysis
Western blot analyses were carried out as described previously [5]. Protein lysates were subjected to immunodetection with antibodies to phosphorylated and then total Akt (Cell Signaling Technology, Beverly, MA, USA), phosphorylated, and then total IB (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-B DNA binding activity was measured in the nuclear protein extracts by electrophoretic mobility shift assay (EMSA), as described earlier [9]. A double-stranded oligonucleotide (NF-
B, 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-
B, and mutant NF-
B 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 I
B (S32A,S36A; a kind gift from Inder M. Verma, The Salk Institute for Biological Studies, San Diego, CA, USA) [9] 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 x 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 5x NF-B 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 5x NF-
B 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 5x NF-
B luciferase reporter in the control cells under serum-starved conditions. Luciferase activity was measured using the Dual Luciferase kit (Promega), as per the manufacturers 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 [5]. For molecular inhibition studies, cells were transiently transfected using FuGene 6 with increasing concentrations of the IB (S32A, S36A) expression plasmid alone, treated with 107 M tamoxifen alone, or transiently transfected with increasing concentrations of the I
B (S32A, S36A) expression plasmid in the presence of 107 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, Students t-test was used.
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Results |
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Discussion |
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NF-B activation has been connected with multiple aspects of oncogenesis, including the control of apoptosis, the cell cycle, differentiation and cell migration (reviewed in [14]). NF-
B is a transcription factor the activity of which is tightly regulated at multiple levels [12, 15]. In most cell types, NF-
B complexes are normally sequestered in the cytoplasm as an inactive complex bound by an inhibitor known as I
B, until a cell is activated by a relevant stimulus [15]. Following cellular stimulation, I
B proteins become phosphorylated by the I
B kinase (IKK). This phosphorylation of I
B results in its ubiquitination and subsequent degradation by the 26S proteasome [12]. The degradation of I
B proteins liberates NF-
B, allowing its translocation to the nucleus, blocking cell-death pathways (reviewed in [16]). Recent studies have now suggested the NF-
B activation is an important mechanism for chemotherapy resistance, and that inhibition of NF-
B significantly enhances tumor cell response to chemotherapeutic agents [17].
Activation of the Akt pathway has been reported to stimulate IKK-dependent IB degradation and nuclear translocation of NF-
B [18]. In our studies, we observed a correlation between Akt activity and the phosphorylation of I
B
, suggesting that in our model, regulation of NF-
B is at least in part through Akt regulation of I
B degradation. Studies are ongoing to delineate the exact mechanisms by which Akt modulates NF-
B activity. However, although the mechanism(s) remains to be determined, it is clear from our studies, as well as those done in other laboratories [19], that Akt activation of NF-
B 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-B activity by inhibiting I
B
degradation, correlating with antitumor activity against both solid and hematologic tumor types [20]. 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 [21, 22], 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-
B 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-B may be an important mechanism in the development of tamoxifen-resistant breast cancer. Several studies have also now demonstrated that NF-
B plays a role in blocking the efficacy of cancer chemotherapies and radiation [16]. Activation of NF-
B 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 [23]. Our data, in which we found that the use of NF-
B inhibitors restores tamoxifen response in the tamoxifen-resistant cells, indicate that a combination regimen of tamoxifen with NF-
B inhibitors may have great efficacy in a subset of resistant breast cancers.
However, there are concerns regarding the targeting of NF-B for cancer treatment. These concerns range from the fact that current NF-
B inhibitors are not specific to issues regarding the potential proapoptotic and critical immunological functions of NF-
B. Moreover, the role of NF-
B could be different in different tumor types. Although rare, there are systems in which NF-
B has been shown to play a proapoptotic role in addition to its more common antiapoptotic role [24]. These concerns strongly indicate that further studies are necessary to dissect the roles of NF-
B in a variety of cancers, and to determine the applicability of inhibiting NF-
B as an adjuvant approach in standard approaches to cancer therapy. For this reason, further investigation is necessary into the role of NF-
B activation through the Akt pathway in the development of hormone refractory breast cancer, and the potential use of agents that target NF-
B as an effective treatment strategy for this relatively resistant disease.
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Acknowledgements |
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FOOTNOTES |
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