The macrophage inhibitory cytokine integrates AKT/PKB and MAP kinase signaling pathways in breast cancer cells
Wyatt Wollmann 1,
Mike L. Goodman 1,
Poornima Bhat-Nakshatri 4, 5,
Hiromitsu Kishimoto 1,
Robert J. Goulet, Jr 1,
Sanjana Mehrotra 3,
Akira Morimiya 3,
Sunil Badve 3 and
Harikrishna Nakshatri 1, 2, 4, 5, *
1 Department of Surgery, 2 Department of Biochemistry and Molecular Biology, 3 Department of Pathology and 4 Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA and 5 Walther Cancer Institute, Indianapolis, IN 46208, USA
* To whom correspondence should be addressed at: R4-202 Indiana Cancer Research Institute, 1044 West Walnut Street, Indianapolis, IN 46202, USA. Tel: +1 317 278 2238; Fax: +1 317 274 0396; Email: hnakshat{at}iupui.edu
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Abstract
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Macrophage inhibitory cytokine 1 (MIC-1), a divergent member of the transforming growth factor beta superfamily, plays a role in the progression of a number of cancers, including breast, gastric, prostate and colorectal carcinomas. Serum MIC-1 levels are elevated in patients with metastatic prostate, breast and colorectal carcinomas. In vitro studies have revealed a cell type-specific role for MIC-1 in senescence and apoptosis. MIC-1 activates the survival kinase AKT/PKB in neuronal cells. Depending on the cell type, it activates or represses the MAP kinases ERK1/2. Mechanisms responsible for an increased MIC-1 expression in cancers and the consequences of MIC-1 overexpression, however, are not known. In this study, we show that AKT/PKB directly regulates the expression of MIC-1 in breast cancer cells. Sequences within 88 to +30 of the MIC-1 promoter are required for the AKT-mediated induction of MIC-1. This region of the promoter contains two SP-1 binding sites (SP-1B and SP-1C), which bind to the SP-1 and SP-3 proteins. Mutation of SP-1C but not SP-1B reduced the AKT-mediated activation of MIC-1. MIC-1 increased the basal ERK1 phosphorylation and prolonged the estrogen-stimulated ERK1 phosphorylation in MCF-7 breast cancer cells without altering the phosphorylation status of AKT/PKB. Immunohistochemistry with MIC-1 antibody revealed an MIC-1 expression within the cancer cells of primary breast cancer and in the MCF-7 xenografts. Furthermore, a limited analysis of RNA from primary breast cancers revealed an overexpression of MIC-1 in tumors, compared with normal tissues. These results suggest that AKT/PKB through MIC-1 could regulate the ERK1 activity and the MIC-1 expression levels may serve as a surrogate marker for the AKT activation in tumors.
Abbreviations: CA-AKT, constitutively active AKT; EMSA, electrophoretic mobility shift assay; ER
, estrogen receptor alpha; KD-AKT, kinase dead AKT; MIC-1, macrophage inhibitory cytokine 1; RTPCR, reverse transcriptionpolymerase chain reaction
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Introduction
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The serine/threonine kinase AKT/PKB is a major cell survival kinase for a number of cell types, including breast cancer cells (1). It is activated by growth factors, including epidermal growth factor, platelet derived growth factor and heregulin (1). Constitutive activation of AKT is observed in a number of cancers, including breast cancer (24). Constitutively active AKT (CA-AKT) along with telomerase can immortalize mammary epithelial cells (5). In transgenic mouse models, CA-AKT alone induces hyperplasia and collaborates with ErbB2 to induce mammary carcinogenesis (6,7). It is also suggested that AKT/PKB phosphorylates Raf kinase in breast cancer cells leading to the inhibition of Raf-MEK-ERK signaling and shifting of the cellular response from cell cycle arrest to proliferation (8,9).
We recently reported the AKT/PKB-mediated activation of estrogen receptor alpha (ER
) and tamoxifen resistance in MCF-7 cells (10). MCF-7 cells engineered to overexpress CA-AKT showed an
5-fold increase in the expression of macrophage inhibitory cytokine (MIC-1). MIC-1 (also known as NAG-1, GDF-15, PDF, PLAB and PTGFB) is a divergent member of the transforming growth factor superfamily and is considered as a biomarker for the p53 pathway activation (1113). Although in vitro studies suggested anti-proliferative and pro-apoptotic functions of MIC-1 in breast, prostate, colon cancers as well as in glioblastomas, analysis of serum samples of patients showed a striking correlation between elevated MIC-1 levels and the metastatic progression of colorectal, breast and prostate cancers (1421). The MIC-1 level is also elevated in the serum of patients with pancreatic ductal adenocarcinoma (22). MIC-1 increases the invasiveness of gastric cancer cells by upregulating the expression of urokinase plasminogen activator (23). Elevated levels of serum MIC-1 is associated with cardiovascular events, whereas lower levels of MIC-1 is a predictor for miscarriage in women (24,25). MIC-1 is also overexpressed in cancer cells undergoing senescence in response to chemotherapeutic treatment (26). These pleiotropic effects of MIC-1 resemble that of TGFß, which is considered as a tumor suppressor during the early stages of cancer and a growth/metastasis enhancer in the later stages of cancer (27).
This study was initiated to understand the role of AKT in regulating the MIC-1 expression and to delineate the consequences of MIC-1 expression on basal and inducible ERK1/2 and AKT activity in breast cancer cells. We show that AKT directly increases the MIC-1 expression in breast cancer cells through an SP-1 binding site. We also show that recombinant MIC-1 increases the basal ERK1 phosphorylation and prolongs estrogen-induced ERK1 phosphorylation. These results suggest that MIC-1 serves as an integrator of the AKT and ERK pathways in breast cancer cells.
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Materials and methods
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Breast cancer cells
MCF-7 and ZR-75-1 breast cancer cells were purchased from American Type Tissue Culture Collection (Manassas, VA) and maintained in MEM plus 10% fetal calf serum.
Recombinant plasmids and transient transfection
The MIC-1 promoter corresponding to the nucleotides 966 to +30 was amplified from the human genomic DNA by PCR and the amplified product was cloned into the chloramphenicol acetyl transferase reporter vector pBL-CAT3+ (28). The MIC-1 promoter sequences were verified by sequencing. Promoter deletion mutants were constructed by PCR. SP-1 mutants of MIC-1 promoter were constructed using a single-step mutagenesis kit from Stratagene (Cedar Creek, TX). Cells were harvested 48 h after transfection and a CAT assay was performed as described previously (10). CA-AKT and kinase dead AKT (KD-AKT) constructs have been described previously (10). MCF-7 cells overexpressing CA-AKT were generated using the bicistronic retrovirus vector pQXIN (BD Sciences, Palo Alto, CA). The CAT assays were performed three to six times.
Western blot analysis
Cell lysates were prepared in a radioimmunoassay buffer and subjected to western analysis as described previously (10). Phospho-AKT (S473), AKT, phospho-ERK and ERK antibodies were purchased from Cell Signaling (Beverly, MA). Recombinant heregulin ß1 and MIC-1 were purchased from R&D systems (Minneapolis, MN).
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using nuclear extracts from MCF7pQXIN and MCF-7CA-AKT cells. The preparation of the nuclear extract and EMSA conditions have been described previously (29). In competition assays, the nuclear extracts were incubated with an unlabeled SP-1 consensus sequence containing oligonucleotides or 88 to +32 region of the MIC-1 (50-fold access) for 10 min on ice before the addition of labeled 88 to +32 region of the MIC-1 promoter. In supershift assays, nuclear extracts along with the probe was incubated with SP-1 or SP-3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 10 min before electrophoresis.
Immunohistochemistry
Sections of 4 µ thickness were de-paraffinized and hydrated. Antigen retrieval was performed in 140 mM citrate buffer, pH 6.0 in a de-cloaking chamber (Biocare) at 125°C for 5 min. The slides were then allowed to cool for 20 min at room temperature. The endogenous peroxidase activity was blocked by Peroxo-Block (Zymed) for 2 min. The slides were then incubated with 2% gelatin for 30 min to decrease the non-specific staining followed by blocking the endogenous avidinbiotin activity by AvidinBiotin Blocking kit (Vector Laboratories). The slides were incubated with 1:100 goat polyclonal MIC1 antibody (R&D systems) for 1 h at room temperature. For the subsequent steps, the avidinbiotinperoxidase method with a Vectastain ABC Elite kit (Vector Laboratories) was used according to the manufacturer's instructions. The stain was visualized using Dako liquid DAB plus substrate chromogen solution and hematoxylin QS (Vector Laboratories) counter stain.
Analysis of MIC-1 expression in primary breast cancer by RTPCR
Total RNA from tumor tissues or the adjoining normal tissue was isolated by guanidinium isothiocyanate method. RTPCR was performed using the single step RTPCR kit from Invitrogen (Carlsbad, CA). The primers used were 5'-CGCTCCGCGCGTCGCTGGAAG-3' and 5'-GGAGCGACTCCCCGGTGTCGG-3' for MIC-1; 5'-TGGAGAAACTGCTGCCTCAT-3' and 5'-GGAGATGTTGAGCATGTTCA-3' for 36B4.
Statistical analysis
Data were analyzed using GraphPad software (Graphpad.com). Analysis of variance was employed to determine the P-values between mean measurements. A P-value of <0.05 was deemed significant. Error bars on all histograms represent a standard deviation between the measurements from 35 experiments.
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Results
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MIC-1 expression is elevated in breast cancer
We have previously shown an elevated expression of MIC-1 in MCF-7 breast cancer cells overexpressing CA-AKT. These cells are also resistant to tamoxifen. To determine whether MIC-1 is overexpressed in primary breast cancers, we performed a semiquantitative RTPCR of RNA isolated from primary breast cancers and adjacent normal tissue. MIC-1 transcript levels were elevated in primary breast cancer samples when compared with the adjoining normal tissues (Figure 1A). To investigate whether MIC-1 is expressed in cancer cells or stromal cells, we performed an immunohistochemistry of primary tumor samples. Only cancer cells expressed MIC-1 (Figure 1B).

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Fig. 1. MIC-1 expression in primary breast cancer. (A) MIC-1 transcripts in breast cancer homogenate as well as in adjoining tissues of some tumors were measured by RTPCR (35 cycles). The integrity and equal quantity of RNA in each reaction were verified by RTPCR using primers specific to the housekeeping gene 36B4 (20 cycles). (B) Immunohistochemistry reveals the expression of MIC-1 in cancer cells but not stromal cells. (C) MIC-1 is expressed in tumors derived from MCF-7 cells in nude mice.
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We used MCF-7 cell derived tumors in nude mice to evaluate whether the stromalcancer cell interaction plays a role in the MIC-1 expression. These tumors were obtained from animals with slow-release estrogen pellets. MCF-7 cells form tumors in nude mice only on supplementation with estrogen (30). MCF-7 cells maintained in vitro do not express MIC-1 (10). However, MIC-1 was readily detectable in the MCF-7 xenografts (Figure 1C). Thus, it appears that a stromal:cancer cell interaction contributes to the MIC-1 expression in cancer cells.
AKT directly increases MIC-1 expression
As our previous studies have shown a role for AKT in MIC-1 expression and AKT is activated in breast cancer owing to the autocrine and paracrine activity of growth factors, we examined whether AKT directly regulates the MIC-1 promoter activity. Previous studies have identified several regulatory elements in the MIC-1 promoter (28). The basal transcription requires three binding sites for SP-1/SP-3 transcription factors (SP-1A, SP-1B and SP-1C), which are located between 133 and +41 region of the promoter. SP-1B is a perfect SP-1 binding site (GGGCGG) and binds preferentially to SP-1 and SP-3, whereas SP-1C is an imperfect SP-1 binding site (AGGCGG) and binds poorly to SP-1 and SP-3. However, it binds strongly to a truncated SP-3 produced through an internal translation initiation (28). To determine whether AKT utilizes any of these regulatory elements to increase the MIC-1 expression, we transfected MCF-7 breast cancer cells and LnCAP prostate cancer cells with MIC-1/CAT reporter with or without a CA-AKT or KD-AKT expression vector (10). CA-AKT, but not KD-AKT, increased the MIC-1/CAT expression in both cell types (Figure 2A). A series of deletion mutants were made to localize the region of the MIC-1 promoter required for an AKT-mediated activation. Although deletions affected the basal activity of the promoter, the AKT-mediated activation was not significantly affected by deletions. A reporter construct with only 88 to +30 of the MIC-1 promoter (88 + 30/CAT), which contains SP-1B and SP-1C sites, was induced by CA-AKT (Figure 2B). Thus, the AKT responsive element is located within 88 to +30 of the MIC-1 promoter, which has been reported previously to be important for the basal expression of the gene (28).

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Fig. 2. AKT increases MIC-1 expression. (A) CA-AKT but not KD-AKT increases the MIC-1 promoter/reporter activity in MCF-7 cells. MCF-7 cells were transfected with a CAT reporter containing sequences 966 to +30 of the MIC-1 promoter (10 µg) with or without CA-AKT or KD-AKT. The empty expression vector pCDNA3 (0.5 µg) was included in the control. The transfection efficiency was measured using the RSV-ß-galactosidase expression vector (2 µg). CAT activity in equal number of ß-galactosidase units was measured 48 h after transfection. CA-AKT increased the MIC-1 promoter activity in LnCAP cells. (B) The AKT response element is located within the 88 to +30 region of the MIC-1 promoter. A series of deletion MIC-1 promoter constructs were transfected into MCF-7 cells with or without CA-AKT as above and CAT activity was measured 48 h after transfection. It is noted that CA-AKT had no effect on the residual expression of pBL-CAT3+ reporter, which lacks any enhancer/promoter region, as well as pBL-CAT2+ reporter, which contains an HSV TK promoter but not an enhancer (data not shown).
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SP-1C element is required for AKT-mediated activation of MIC-1
To determine the requirements of SP-1 motifs in the AKT-mediated activation of MIC-1 promoter, we mutated SP-1B (MIC1/SP-1Bmut) or SP-1C (MIC1/SP-1Cmut) sites in the 88 + 30/CAT reporter. The basal activity of MIC-1/SP-1Bmut was similar to that of 88 + 30/CAT and AKT increased its activity (Figure 3). In contrast, MIC-1/SP-1Cmut CAT was not efficiently induced by AKT. These results suggest that AKT increases the MIC-1 promoter activity through the SP-1C site.

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Fig. 3. SP-1C element is required for AKT-mediated activation of MIC-1. The 88/+30 MIC-1 CAT reporter with mutation in SP-1B (MIC-1/SP-1Bmut) or SP-1C (MIC-1/SP-1Cmut) were co-transfected with either the empty vector pcDNA3 or CA-AKT. CAT activity was measured as in Figure 2.
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AKT does not alter transcription factor binding to the 88 to +30 region of MIC-1 promoter
To determine whether AKT alters the binding of transcription factors to the 88 to +30 region, we first generated MCF-7 cells overexpressing CA-AKT (MCF-7CA-AKT) by a retrovirus-mediated gene transfer. Nuclear extracts from cells with the vector alone (MCF-7pQXIN) or CA-AKT were used for EMSA with 88 to +30 as a probe. A similar DNA binding pattern was observed in both the cell types (Figure 4A). The intensity of complex I varied from experiment to experiment (compare Figure 4A and B). Unlabeled oligonucleotide containing a consensus SP-1 binding site reduced the intensity of this complex I, suggesting that this complex corresponds to the SP-1:DNA complex (Figure 4B). Interestingly, a competition with the unlabeled SP-1 consensus oligonucleotide increased the binding of a minor slow mobility complex (indicated by asterisk).

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Fig. 4. AKT does not alter transcription factor binding to 88 to +30 region of MIC-1 promoter in vitro. (A) EMSA was performed with nuclear extracts from MCF-7pQXIN (control) or MCF-7CA-AKT cells. The 88 to +30 region of the MIC-1 promoter was used as the probe. The intensity of complex I was reduced by an unlabeled oligonucleotide containing a consensus SP-1 binding site identical to SP-1B element. An asterisk indicates a minor complex whose intensity increased upon competition with unlabeled SP-1 consensus oligonucleotide. (B) Binding of SP-1 and SP-3 to 88 to +30 region of MIC-1 was determined by antibody supershift assays. While SP-1 antibody disrupted complex I, SP-3 antibody supershifted complex II, III and the minor complex. SS, supershift.
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We used antibody supershift assays to determine whether SP-1 or SP-3 binds to 88 to +30 region. The SP-1 antibody disrupted complex I, whereas complex II and III as well as the minor complex (corresponding asterisk in Figure 4A) were supershifted by SP-3 (Figure 4B). Taken together, these results indicate that AKT alters the activity but not binding of a transcription factor(s) to the 88 to +30 region of the MIC-1 promoter. SP-1 and SP-3 appear to be the major proteins that bind to this region of the promoter.
MIC-1 increases ERK-1 phosphorylation in MCF-7 and ZR-75-1 cells
ERK pathways play a distinct role in the growth and differentiation of breast cancer cells. For example, a transient activation of Raf-MEK-ERK along with the activation of AKT pathway by insulin like growth factor leads to the proliferation of MCF-7 cells (31,32). However, a prolonged activation of the Raf-MEK-ERK cascade leads to the growth inhibition of these cells. The PI3 kinase/AKT pathway helps in the transient Raf-MEK-ERK activation by downregulating the Raf activity through a phosphorylation of S259 (8,9). Anti-estrogens such as tamoxifen has been shown to induce proliferation instead of inhibiting the growth of MCF-7 cells with an elevated basal ERK activity (33). A recent report suggested that MIC-1 activates the PI3 kinase/AKT but represses the ERK pathway in neuronal cells (34). In gastric cancer cells, however, MIC-1 induces the ERK pathway (23). These contrasting cell type-specific effects of MIC-1 on ERK prompted us to investigate the effect of MIC-1 on basal, estrogen and heregulin ß1-inducible ERK and AKT activation in MCF-7 cells. Contrary to neuronal cells, MIC-1 increased the basal ERK phosphorylation (Figure 5A). ERK1 is the major active ERK in MCF-7 cells. Estrogen induced ERK1 in a biphasic manner in the absence of MIC-1, whereas ERK1 activation was continuous in cells treated with both estrogen and MIC-1. MIC-1 had a modest effect on the heregulin ß1-induced ERK activation (Figure 5A). We next examined the effect of MIC-1 on AKT activation. MIC-1 had a minimum effect on the basal or inducible AKT activation.

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Fig. 5. The effect of MIC-1 on ERK and AKT activity in MCF-7 and ZR-75-1 cells. (A) MIC-1 increases phospho-ERK1 levels in MCF-7 cells. MCF-7 cells, serum starved for 48 h, were incubated with recombinant MIC-1 (25 ng/ml) for 1 h followed by estrogen (0.1 nM) or heregulin ß1 (50 ng/ml) for the indicated time. Activated ERK and AKT levels were measured using phospho-specific ERK and phospho-AKT (S473) antibodies, respectively. The same blot was reprobed with antibodies that measure total ERK or AKT. It is noted that MIC-1 increased the basal phospho-ERK levels as well as caused a sustained instead of biphasic ERK activation in estrogen-treated cells. MIC-1 had only a modest effect on heregulin ß1-induced ERK. (B) MIC-1 increases ERK1 phosphorylation in ZR-75-1 cells and modulates the estrogen-mediated changes in the phospho-ERK1 and phospho-ERK2 ratio.
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We determined the effect of MIC-1 on ERK activation in ZR-75-1 cells to ensure that the observed effects are not specific to MCF-7 cells. Unlike in MCF-7 cells, both ERK1 and ERK2 are active in these cells to a similar extent (Figure 5B). Surprisingly, estrogen reduced the phospho-ERK2 levels and altered the ratio between phospho-ERK1 and phospho-ERK2 in favor of phospho-ERK1. MIC-1 pre-treatment accelerated an estrogen-induced shift in the phospho-ERK1 and phospho-ERK2 ratio. Neither estrogen nor MIC-1 altered the overall levels of ERK1 and ERK2. As with MCF-7 cells, MIC-1 had no effect on the phospho-AKT levels. From these results, we conclude that MIC-1 enhances the phosphorylation status of ERK protein with a specific effect on ERK1. Moreover, the effect of MIC-1 on ERK is not negatively regulated by AKT.
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Discussion
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In this report, we show a crosstalk between MIC-1, ERK and AKT in breast cancer cells. While AKT increased the MIC-1 expression, MIC-1 elevated the ERK activation. MIC-1, either alone or in combination with estrogen, increased the phosphorylation of ERK1 primarily. The role of ERK in the growth and differentiation of mammary epithelial cells is still not clear. Although initial studies indicated a role for ERK in the proliferation of mammary epithelial cells, its activity has been shown to be required for the differentiation of breast cancer cells by agents such as TPA (31). MCF-7 cells with an elevated basal ERK activity have been shown to be growth-stimulated by tamoxifen (33). AKT pathway, on the other hand, is considered to be a major survival and proliferation pathway for mammary epithelial cells (8,35). It is suggested that AKT blocks the differentiation of mammary epithelial cells by inhibiting ERK (8). The negative crosstalk between AKT and ERK may be abrogated in cells that overexpress MIC-1 because MIC-1 can induce ERK without reducing AKT and MIC-1 is induced by AKT.
MIC-1 appears to shift the ratio between active ERK1 and ERK2 in favor of a more active ERK1 (Figure 5). Consequences of such a shift are not known. Although ERK1 and ERK2 perform redundant functions, knockout animal studies have revealed unique functions for ERK1 and ERK2. While ERK2 knockout animals show an embryonic lethality owing to a defective mesoderm differentiation, ERK1 knockout animals are viable and have a minor defect in the development of T cells (36,37). Although there are no differences in the substrate specificity, ERK1 but not ERK2, binds to scaffold proteins such as MP-1, which enhances its enzymatic activity (38). By altering the ratio between ERK1 and ERK2, MIC-1 in combination with estrogen can increase the level of the enzymatically active species of ERK. In this respect, we have observed a higher expression of ERK2 than ERK1 in a majority of breast cancer cell lines (data not shown), which may limit the effect of MIC-1 to only those cell types that express a higher level of ERK1 when compared with ERK2.
AKT increased the MIC-1 expression in prostate cancer cells as well (Figure 2). This is particularly interesting because the AKT activation in prostate cancer is associated with a poor clinical outcome (39). Interestingly, AKT activation in prostate cancer cells leads to a reduced ERK activation, which is similar to the effect of MIC-1 on ERK in neuronal cells (34). MIC-1 overexpression in prostate cancer is correlated with invasiveness and metastasis (40,41). A recent protein profiling of microdissected prostate tissue revealed an overexpression of MIC-1 protein in high-grade prostatic intraepithelial neoplasia and prostate cancer cells of Gleason grade 3 (42). Additional studies are required to confirm whether the AKT action in prostate cancer is mediated through the induction of MIC-1.
The mechanism by which AKT increases the MIC-1 expression is not known. The basal MIC-1 promoter activity is dependent on three SP-1 response elements; SP-1A, SP-B and SP-1C (28). SP-1B is a perfect SP-1 binding site whereas SP-1A and SP-1C are imperfect SP-1 binding sites. SP-1A site is not involved in the AKT-mediated activation of MIC-1 because this site is upstream of the minimum region of the promoter required for the AKT-mediated activation. Furthermore, a reporter containing 966 to +30 promoter region of MIC-1 with SP-1A mutation was activated by AKT (data not shown). SP-1C is involved in the AKT-mediated activation of MIC-1. SP-1 has been previously linked to the AKT-mediated activation of FRA-1 (43). Activation of FRA-1 by AKT involved a transcriptional upregulation and an increased DNA binding of SP-1 (43). However, this is not the case with the MIC-1 promoter because we did not observe an increased SP-1 binding in cells overexpressing CA-AKT. SP-1C binds preferentially to truncated SP-3 (28). We also observed a preferential binding of SP-3 to the MIC-1 promoter region that respond to AKT (Figure 4B). We believe that AKT increases the interaction of SP-3 bound to the SP-1C site with transcriptional co-activators or other transcription factors. One such candidate is ER
, which indirectly activates the promoters through an interaction with SP-1 (44) and is phosphorylated by AKT (10). In our previous study, we have shown an estrogen-dependent increase in MIC-1 in MCF-7 cells overexpressing CA-AKT (10). In addition, a number of co-regulators are phosphoproteins and some of them may be the target of AKT (45,46). COUP-TF nuclear receptors have been previously shown to increase the MIC-1 expression through SP-1 binding sites (28). However, COUP-TFs are not likely to be the target of AKT because they lack the consensus AKT phosphorylation sites and we did not observe a synergistic increase in the MIC-1 expression upon co-expression of COUP-TFs and AKT (data not shown). It is also noted that both SP-1 and SP-3 lack the consensus AKT phosphorylation sites.
The role of MIC-1 in cancer progression is unclear if not controversial. We observed the MIC-1 expression in primary breast cancer and a recent study showed the rapid induction of MIC-1 in cancer cells subsequent to a neoadjuvant therapy (47). MIC-1 has been linked to the senescence and apoptosis of cancer cells (18,26). It is also overexpressed in cancer cells, which are sensitive to chemotherapy, but not in cancer cells that are resistant to chemotherapy (26). In contrast to these pro-cell death function, MIC-1 overexpression is linked to the progression of gastric cancer (23). This paradoxical role of MIC-1 in cancer could be related to its ability to induce ERK. Depending on the cell type and duration of induction, ERK can induce cell cycle arrest, senescence or drug resistance (33,48,49).
Thus, in cancer types in which ERK induces a growth arrest or senescence, MIC-1 may play a pro-death role. Similarly, MIC-1 expression may be linked to a sensitivity to chemotherapy in cancers where chemotherapy-induced cell death requires an ERK activation (50). Rapidly proliferating cells are more sensitive to chemotherapy compared with quiescent cells. MIC-1 may sensitize certain cancer cells to chemotherapy by increasing their proliferation. In cancers where ERK activation is linked to a survival through the AP-1 pathway, MIC-1 may play a role in the progression of cancer. MIC-1 mediated upregulation of urokinase plasminogen activator in gastric cancer cells could be a consequence of the AP-1 activation (23). With respect to breast cancer, we suspect that MIC-1 plays a role in cancer progression, particularly in ER
-positive breast cancers because it induces ERK without reducing AKT. Both ERK and AKT modulate ER
activity (51). We observed an MIC-1 expression in MCF-7 cell-derived tumors in nude mice (Figure 1C), which suggests a correlation between proliferation and MIC-1 expression. Elevated MIC-1 levels in the serum of patients with metastatic breast cancer may indicate the rate of proliferation of cancer cells at sites of metastasis (21). Additional immunohistochemical studies with tissues obtained from the sites of metastasis are required to test this possibility.
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Acknowledgments
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This work is supported by Public Services Award CA-89153 from the National Cancer Institute (to H.N.).
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Received September 1, 2004;
revised January 10, 2005;
accepted January 18, 2005.