Background
Metastatic disease is the main cause of death in cancer patients. The interaction of tumor cells with local stroma plays a critical role in metastatic dissemination and determines metastasis organotropism. Identification of the genes providing cancer cells with the abilities to disseminate to specific organs is essential for targeting metastatic cells to improve patient survival.
The c-Myb protein is a transcription factor that plays a key role in regulating the proliferation/differentiation of progenitor cells in bone marrow, colonic crypts, and neurogenic niches [
1]. c-
myb was originally identified as a cellular homolog of v-
myb, the transforming retroviral oncogene linked to avian leukemia [
2]. Since then, c-
myb has been characterized as an oncogene in several human tumor types [
3‐
7], including breast cancer [
8‐
10]. The role of c-Myb in stimulating cell proliferation, suppressing differentiation, and apoptosis is well established [
1,
11]. However, only a few reports concerning the role of c-Myb in controlling tumor invasion have been reported. First, genes coding for some of the proinvasive factors, such as CXCL12 and CXCR4, were identified as c-Myb targets [
12‐
14]. Second, c-Myb is involved in the regulation of epithelial-to-mesenchymal transition (EMT) and invasion in neuroblastoma, colon carcinoma, and embryonic kidney cells through the upregulation of the transcription repressor Slug [
15]. Third, in hepatocellular carcinoma cells, c-Myb promotes cell invasion via the upregulation of osteopontin [
16]. Interestingly, the depletion of endogenous c-
myb in MCF7 breast cancer cells increased tumorigenesis
in vitro and
in vivo, suggesting that c-Myb acts as a tumor suppressor in breast cancer [
17]. However, how c-Myb controls breast carcinoma progression and dissemination remains unresolved. To gain insight into this process, we generated variants of mammary carcinoma cell lines overexpressing c-
myb and investigated their migratory/invasive and metastatic capabilities. We demonstrated that c-Myb regulates the invasive behavior of breast cancer cells in a matrix-dependent manner, possibly via a novel signaling axis causing the deregulation of MMP1, MMP9, and cathepsin D expression.
Discussion
In this report, we identified the regulation of MMP1/9 and cathepsin D by c-Myb as a novel mechanism of the matrix-specific breast cancer cell invasion. c-Myb has been recently identified as a regulator of tumor cell motility and invasion, and the Slug transcription factor was described as the mediator of the c-Myb-induced mesenchymal-like phenotype in neuroblastoma, colon carcinoma, and embryonic kidney cells [
15]. The TGFβ-induced EMT and invasion of estrogen receptor-positive breast cancer cells was also found to be dependent on c-Myb expression [
28]. In our study, we confirmed the regulatory role of c-Myb in control of migration and invasion of breast carcinoma cells as well. We also observed upregulation of Slug in the MYBup variants of MDA-MB-231 cells, but we did not detect any c-Myb-dependent changes in expression of either epithelial or mesenchymal cell markers, such as E-cadherin, N-cadherin, or vimentin (Additional file
5). This may presumably result from the mesenchymal-like phenotype of the parental MDA-MB-231 cells. We demonstrated that c-Myb activates transcription of
cathepsin D in a Slug-independent manner (Additional file
6). This implies that EMT is not an exclusive mode of c-Myb control over cell migration/invasion. In hepatocellular tumor cells, c-Myb stimulated migration/invasion via osteopontin secretion [
16]. Activation of the osteopontin promoter by c-Myb was also reported in melanomas [
29]. However, expression of the osteopontin gene was not deregulated in the c-
myb-overexpressing MDA-MB-231 cells (data not shown) implying that c-Myb uses another mechanism to control migration/invasion of breast cancer cells.
There are two main types of ECM in vertebrates: BM and the stromal/interstitial matrix. Components of the BMs include collagen IV, laminin, perlecan, and nidogen. Stromal/interstitial matrices that form the majority of the body connective tissues are composed primarily of fibrillar collagen I. As both BM and stromal matrices represent steric barriers to cell migration, matrix remodeling is a critical prerequisite for metastasis formation. BM extract/Matrigel is frequently used to assess cell invasive capacity
in vitro. Matrigel invasion
in vitro is considered to simulate the penetration of BMs underlying epithelial cells or blood vessels by tumor cells
in vivo. We documented that c-Myb induces the invasion of MDA-MB-231 cells through Matrigel. Conversely, cancer cell invasion through stromal/interstitial matrices composed primarily of fibrillar collagen I was found to be critical for metastasis in several tumor types [
22,
23]. We found that c-Myb does not activate cell invasion through the collagen I barrier. Modulation of cell invasion by ECM components was described previously [
30‐
32]. Different invasion modes reflecting the specificity of substrates may be attributed to the different requirements on proteolytic systems [
22,
33]. Therefore, we analyzed the expression of candidate proteases in c-
myb-overexpressing and control MDA-MB-231 cells and confirmed the differential expression of
MMP1,
MMP9, and
cathepsin D. We confirmed the recent observation by Bhattarai that the c-Myb upregulates MMP9 in breast cancer cells [
34]. Our demonstration that c-Myb modulates MMP1 has not been described yet. While cathepsin D and MMP9 were upregulated, the expression of MMP1 was considerably reduced in the MYBup variants. c-Myb-induced Matrigel invasion is sensitive to the broad spectrum MMP inhibitor GM6001 (Ilomastat; Additional file
3). As MMP9 is the only MMP found to be upregulated in MYBup cells and collagen IV, the major component of the BM/Matrigel, is the main substrate for MMP9, we hypothesize that apart from cathepsin D, the MMP9 is an effector of the c-Myb-induced Matrigel invasion. MYBup cells exhibiting high MMP9 activity could transverse the Matrigel matrices more efficiently than controls, even in the absence of MMP1. MMP1 (interstitial collagenase) predominantly cleaves collagen I; therefore, the lack of MMP1 in MDA-MB-231MYBup cells might compromise their penetration through the collagen I matrix. Cathepsin D might secure partial invasion of these cells to collagen I substrates [
35], as shown by reduced collagen I penetration of MDA-MB-231MYBup cells with silenced cathepsin D expression (Figure
5).
We propose cathepsin D as a novel downstream mediator of the c-Myb migration/invasion-promoting function. Cathepsin D was suggested as one of the c-Myb-target genes in MCF7 breast cancer cells previously [
36]. Our results confirmed this observation in MDA-MB-231 cells. We identified multiple putative Myb-binding sites in the human
cathepsin D gene promoter using TESS software (Additional file
7). There are conflicting results regarding participation of the cathepsin D in the control of breast cancer cell invasion and metastasis [
37‐
44]. Johnson et al. observed that breast cancer cell invasion did not reflect various release rates of cathepsin D from different subclones of MCF7 cells [
41]. Similarly, Glondu et al. modulated cathepsin D expression using antisense inhibition without any effect on invasiveness of breast cancer cells
in vitro [
42]. In contrast, suppression of cathepsin D by antisense oligonucleotides and shRNA in MCF7 and MDA-MB-231 cells, respectively, associated with reduction of their invasion through Matrigel was observed by others [
43,
44]. The effects of siRNA-mediated silencing of cathepsin D in MDA-MB-231MYBup cells described in our study provide further support to the studies documenting the regulatory function of cathepsin D in breast cancer cell migration and invasiveness. Several hypotheses have been raised concerning the mechanism how cathepsin D exerts its effects in tumors. They include facilitated release of growth factors, degradation of the extracellular matrix to permit invasion of the tumor cells and proteolytic activity-independent stimulation of the tumor cells via protein-binding activity of cathepsin D. We observed that unlike siRNA-mediated suppression of cathepsin D expression, inhibition of its activity using pepstatin A blocked neither migration nor invasion of MDA-MB-231MYBup cells (data not shown). This implies that it is the protein-binding activity of cathepsin D that may be involved in stimulation of the tumor cells. Our results correspond with studies documenting that catalytically inactive mutants of cathepsin D stimulate cell invasion [
45,
46]. Recently, the LRP1 cell surface receptor was identified as a binding partner for pro-cathepsin D in fibroblasts [
47]. Breast cancer cells express LRP1 [
48] and the LRP-induced stimulation of cancer cell motility and invasion was described elsewhere [
49,
50]. Therefore, we can hypothesize that cathepsin D enhances migration/invasion of breast cancer cells via LRP1 signaling.
Despite the upregulation of MMP9 mRNA/protein observed in the cells overexpressing c-
myb, transient transfection studies demonstrated no transactivation of the reporter gene derived from the MMP9 promoter by c-Myb. Similarly, c-Myb did not repress transcription from the MMP1 promoter, although the level of MMP1 decreased in the MYBup cells. c-Myb was demonstrated to act as a transcriptional transactivator/repressor through specific binding to the promoter regions of target genes in numerous studies. However, there are also reports demonstrating that c-Myb can affect gene expression via indirect mechanisms [
51,
52]. We studied the stability of the
MMP9/1 transcripts using actinomycin D and found that c-Myb does not affect the stability of
MMP9/1 mRNAs (data not shown). We hypothesize that structural and functional differences between transiently transfected plasmid DNA and genomic templates, such as inefficient chromatinization, might explain why c-Myb failed to transactivate/repress the MMP9/1 promoters [
53]. There are reports indicating that the Myb-induced chromatin binding and remodeling are essential for the transactivation of its target genes [
54,
55].
We demonstrated that murine c-Myb regulates migration/invasion of mouse 4T1 cells
in vitro. 4T1 cells were employed as orthotopic mammary tumor model because they effectively metastasize and display metastatic characteristics similar to those observed in cancer patients [
56]. Surprisingly, the c-
myb-overexpressing 4T1 cells injected into the mammary fat pads of BALB/c mice exhibited delayed tumor growth and no formation of spontaneous pulmonary metastases. Previous studies described that the
myb genes can function either as oncogenes or as tumor suppressors in different cellular contexts [
57] and there are conflicting results concerning the c-
myb function in breast cancer. Oncogenic role of c-Myb was documented by c-
myb knockdown in estrogen receptor (ER)-positive breast cancer cell lines resulting in block of estrogen-dependent proliferation [
58] and TGFβ-induced invasiveness
in vitro [
28]. Miao et al. have recently published data documenting that established human breast cancer xenografts do not advance when c-Myb is knocked down using shRNA [
59]. The tissue-specific deletion of c-
myb also interferes with mammary tumorigenesis in mouse mammary tumor virus (MMTV)-NEU and MMTV-PyMT mice [
59]. On the other hand, there is also report that c-
myb depletion increases the cell growth and tumorigenesis of MCF7 breast cancer cells both
in vitro and
in vivo [
17] documenting that c-Myb can also act as tumor suppressor. The data based on human breast cancer microarray expression analysis
in vivo showed that high c-Myb expression is associated with a good outcome and high differentiation status of the tumors [
17]. Similarly, a unique subgroup of estrogen receptor-positive human breast cancers with 100% overall survival, no metastatic potential, and high c-
myb expression has been described recently [
60]. Our study of ectopic c-
myb overexpression in a mouse orthotopic tumor model supports the view of c-Myb as tumor-suppressor in breast cancer. The c-Myb-controlled expression of Hep27 gene was suggested as a mechanism how c-Myb exerts its tumor-suppressing function in ER-positive breast cancer [
17,
60]. Hep27 inhibits Mdm2 thereby stabilizes p53 [
61]. Therefore, the c-Myb-Hep27-Mdm2-p53 signaling pathway may have functional significance for ER- and p53wt-positive breast cancers cells [
61]. Interestingly, good prognosis of patients with ER-negative basal-like subtype of breast tumors with frequently mutated p53 was also associated with high c-
myb expression [
17]. To date, the function of c-Myb in metastasis formation in mouse models has not been clarified. It was demonstrated, however, that c-Myb promotes the bone marrow homing of leukemic cells [
15]. Our study showed that overexpressed c-Myb can suppress the formation of pulmonary metastases in a mouse model of mammary carcinoma. We propose that interstitial collagenase may be one of the mediators of the tumor- and metastasis-suppressing function of c-Myb in ER-negative breast cancer cells because interstitial collagenase was clearly downregulated in MDA-MB-231MYBup cells (Figure
4), 4T1MYBup cells (Additional file
8) and in the c-
myb- overexpressing tumors (Additional file
8). A number of observations support this hypothesis. Upregulation of MMP1 was previously associated with advanced stages of breast cancer, and it was thus suggested as a predictive marker for the development of invasive disease [
62]. The shRNA-mediated silencing of MMP1 inhibited growth of MDA-MB-231 cells orthotopically implanted into the mammary fat pads of nude mice [
63]. The requirement for MMP1 for breast tumor growth was partly attributed to breakdown of the fibrous stroma within the mammary fat pad and partly to the liberation of growth factors present in ECM [
63]. In addition, the MMP1/protease-activated receptor 1 signaling axis promoting mammary tumor growth and metastasis was identified [
64‐
66]. In addition, Gupta et al. reported that MMP1 silencing in combination with
epiregulin (EREG),
cyclooxygenase 2 (COX2), or
MMP2 delayed tumor progression [
67]. Interestingly,
MMP1,
EREG, and
COX2 are parts of the clinically validated lung metastasis signature [
68]. This signature comprises genes marking and mediating breast cancer metastasis to the lungs.
MMP1 belongs to the family of genes with dual functions conferring both breast tumorigenicity and lung metastagenicity [
68,
69].
Moreover, MMP1 has been implicated in pulmonary extravasation [
67,
70]. The barriers to metastasis are distinct in different organs [
71,
72]. The lung vascular endothelial junctions act as barriers limiting cell passage. In contrast, the bone marrow and liver vasculature consists of capillary vascular channels possessing a discontinuous endothelium [
71,
73]. Therefore, lung metastases may require robust extravasation such as those provided by
MMP1,
EREG,
COX2, and
MMP2 [
67,
71]. In contrast, the requirement for effective extravasation is not principal for bone and liver metastasis [
71,
73,
74]. These findings correspond with our results revealing organ-specific differences in the metastatic ability of c-
myb-overexpressing cells. The deficit of MMP1 function in the c-
myb-overexpressing cells may contribute to the selective disadvantage of these cells in lung colonization. However, they can still initiate metastasis in bone and liver.
We demonstrated that enhanced migration and invasion of c-
myb-overexpressing breast cancer cells through Matrigel
in vitro does not imply increased metastatic capacity. Inconsistency in proinvasive behavior of tumor cells
in vitro and metastatic potential in mouse xenograft models
in vivo was also described previously [
75,
76]. It was postulated that the capacity of tumor cells to metastasize is determined not only by the inherent characteristics of the cancer cells, such as motility and
in vitro invasiveness, but it is also modulated by the cancer cell microenvironment, such as ECM deposition, the presence of other proteases, cytokines, growth factors and adaptor proteins [
75‐
77]. Apparently, there are tissue-specific factors in the host that participate in the control of cancer cell invasiveness [
78].
Methods
Plasmids
Constructs encoding human (pcDNA3-hcMYB) and mouse c-Myb proteins (pcDNA3-mcMYB) were kindly provided by J. Bies [
79]. The reporter construct p6MBSluc containing six Myb-binding sites upstream of luciferase cDNA was kindly provided by T. Nakano [
80]. CMV-βgal was described elsewhere [
81]. Reporter constructs with cathepsin D promoter sequences (2.85 and 0.82 kb) in pGL3 were kindly provided by J. Chirgwin [
82]. Reporter constructs containing the MMP1 promoter sequences (4.3-1 G, 1.606, and 0.624 kb) in pGL3 were kindly provided by C. E. Brinckerhoff [
83] and A. Galloway [
84]. The MMP9 promoter sequence (-1362/+19) was obtained from the genomic DNA of human blood cells by PCR using the forward primer 5'-GCGCGCAGATCTTATAGACCCTGCCCGATGCCGG-3' and the reverse primer 5'-GCGCGCAAGCTTTGGTGAGGGCAGAGGTGTCTGAC-3'. The PCR product was cut with
Bgl II/
Hind III and ligated into pGL2-basic (Promega, Madison, WI).
Cells and transfection
Human MDA-MB-231 and mouse mammary carcinoma 4T1 cells obtained from American Type Culture Collection were cultured in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% FCS, 2 mM L-glutamine 100 U/ml penicillin and 100 μg/ml streptomycin. Medium for 4T1 cells was supplemented with 4500 mg/l glucose and 1 mM sodium pyruvate. MDA-MB-231 cells were transfected with the pcDNA3-hcMYB plasmid and the myb-less control plasmid. 4T1 cells were transfected with the pcDNA3mcMYB plasmid and the myb-less control plasmid. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Transfectants were selected with 800 μg/ml G418 (MDA-MB-231 cells) and 300 μg/ml G418 (4T1 cells) and cloned by limiting dilution.
Transmigration/invasion assay
Cell migration and invasion were analyzed using Cultrex cell invasion assay (Trevigen, Gaithersberg, MD) according to the manufacturer's instructions. Briefly, for the invasion assay, the membrane in the upper chamber of 96-well plate was coated with 0.5 × basement membrane matrix/Matrigel. For the transmigration assay, the membrane was left uncoated. MDA-MB-231 cells were starved in the serum-free medium for 8 hours prior assay, then seeded at a density 5 × 104/well. After 16 h, the cells on the lower surface were dissociated and stained with the Calcein-AM. The amount of emitted fluorescence was determined by Synergy HT microplate reader (Bio-tek, Winooski, VT) to quantify the relative cell migration/invasion.
Kinetics of cell migration/invasion
To monitor cell migration/invasion in real time, we used the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16 (Roche, Indianapolis, IN). The CIM-plate 16 is a 16-well system in which each well is composed of upper and lower chambers separated by an 8-μm microporous membrane. Migration/invasion was measured as the relative impedance change (cell index) across microelectronic sensors integrated into the bottom side of the membrane. For the cell invasion experiments, the membrane was coated with either Matrigel (BD Biosciences, San José, CA) or collagen I (Sigma, St. Louis, MO). Matrigel was diluted 1:40 in serum-free medium and allowed to polymerize at 37°C for 4 h. The collagen solution was neutralized with NaOH and allowed to form a 3D gel at 37°C for 1 h. For the cell migration experiments, the membrane was left uncoated. FCS (10%) was used as a chemoattractant. Cells were starved in a serum-free media for 8 h. Cells (7.5 × 104) were added in duplicates to the upper chambers. Migration/invasion was monitored every 10 or 15 min for several hours. For quantification, the cell index at indicated time points was averaged from at least three independent measurements.
RNA isolation and quantitative RT-PCR
Total RNA was isolated from MDA-MB-231 cells using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA (0.5 μg) was used for first-strand cDNA synthesis with the QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA). Human MMP2, 3, 7, 10, 11, 13 mRNA were quantified by qRT-PCR with specific primers and RT2 SYBR Green/ROX PCR Master Mix (SABiosciences, Frederick, MD). Human c-myb, cathepsin D, MMP9, and MMP1 mRNAs were quantified by qRT-PCR using specific TaqMan probes (Applied Biosystems, Foster City, CA). GAPDH was used as an internal control. All PCR reactions were performed in triplicate for each sample and were repeated at least three times. The qRT-PCR data were analyzed by the ΔΔCt method.
Immunoblotting
Cell lysates were subjected to SDS-PAGE and immunoblotted as previously described [
85]. The conditioned media were concentrated using Amicon Ultra Centrifugal Filters with the Ultracel-10 membrane (Millipore, Billerica, MA). Protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). Equal amounts of total protein (30 μg) were loaded. Blots were probed with anti-Myb (05-175, Millipore, Billerica, MA), anti-cathepsin D (610801, BD Biosciences, San José, CA), anti-MMP1 (1976-S, EpiSelect MMP sampler kit, Epitomics, Burlingame, CA), and anti-MMP9 (G657, Cell Signaling Technology, Beverly, MA) antibodies. To control for sample loading, the blots were probed with a β-actin-specific antibody (A5060, Sigma, St. Louis, MO). Blots were developed by standard ECL procedure using Immobilon Western Chemiluminiscent HRP Substrate (Millipore, Billerica, MA).
Cathepsin D assay
The cells (9 × 105) were collected in 80 μl of sterile-filtered water and subjected to three freeze-thaw cycles. After centrifugation at 25 000 × g for 5 min, 20 μg of cell extracts were subjected to the Cathepsin D Assay (Sigma, St. Louis, MO) according to the manufacturer's instructions. Pepstatin A was added to determine the cathepsin D-specific fluorescence signals. The plates were incubated at 37°C for 30 min, and readings were performed in 2-min intervals using a Synergy HT microplate reader (Bio-tek, Winooski, VT).
Transactivation assay
To determine transactivation by c-Myb, MDA-MB-231 wt, vector, and MYBup cells were transiently cotransfected with p6MBSluc/pGL3-CD/pGL2-MMP9/pGL3-MMP1 and CMV-βgal plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Cells were cultured for 24 h and processed for luciferase and β-galactosidase assays as described elsewhere [
86]. The luciferase activity of each sample was expressed in relative light units and normalized for transfection efficiency according to the β-galactosidase activity.
Cathepsin D/c-myb knockdown using siRNA
The cathepsin D siRNA oligos (5'-GGAUCCCGCUGCACAAGUUTT-3') were purchased from Ambion (Austin, TX). The c-myb siRNA oligos (5'-UAUAGUGUCUCUGAAUGGCUGCGGC-3') were purchased from Invitrogen. ON-TARGETplus nontargeting pool siRNA (Dharmacon, Lafayette, CO) was used as a negative control. Transfection with siRNA was performed using xtremeGENE siRNA transfection reagent according to the manufacturer's recommendations (Roche, Indianapolis, IN). siRNA (30 nM, final concentration) was added to the plates for 3 h. After 72 h, the cells were processed for a cell migration assay as described above. At the same time, the cells were harvested, and cathepsin D/c-Myb expression was determined by immunoblotting.
To evaluate the formation of spontaneous metastases, c-
myb- overexpressing and control 4T1 tumor cells (1 × 10
5 in a 20-μl Matrigel:PBS solution, 1:1) were injected into the fourth mammary fat pad of female BALB/c mice aged 6-8 weeks. The use of these animals followed the European Community Guidelines as accepted principles for the use of experimental animals. The experiments were performed with the approval of the Institutional Ethical Committee. Tumor growth was monitored at least twice a week by measuring the tumor length (l) and width (w) with caliper, and tumor volume was calculated using the equation l × w
2/2. The mice were euthanized when the mean tumor diameter was approximately 1.2 cm [
87]. Tumors were excised and weighed. Lungs were fixed in Bouin's solution, and liver and bones were fixed in 10% buffered formalin. Surface metastatic nodules in lungs were determined by dissecting microscopy. Lungs, liver and bone tissues were processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin (H&E). Bones were decalcified overnight before embedding. The number of pulmonary metastatic lesions was determined by histological examination. Every 10th consecutive section was examined for the presence of metastases. The extent of liver and bone metastases was evaluated semi-quantitatively by light microscopy.
Statistics
Values were expressed as means ± standard deviations. To determine statistical significance, the values were compared by unpaired two-tailed t-test. Differences were considered to be significant at p < 0.05.
Authors' contributions
LK and PB carried out experiments in vitro and analyzed results as well as took part in writing the manuscript. LP participated in performing transactivation assays. MM, LK, KS and ZP participated in in vivo experiments. MH provided histological analysis. JS supervised the experimental work, participated in data analysis and interpretation of results and revised the manuscript. All authors read and approved the manuscript.