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Cell, Tumor, and Stem Cell Biology |
1 Breast Cancer Program, Departments of Oncology and 2 Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland
Requests for reprints: Saraswati Sukumar, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Room 410 CRB, 1650 Orleans Street, Baltimore, MD 21231-1000. Phone: 410-614-2479; Fax: 410-614-4073; E-mail: saras{at}jhmi.edu.
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Abstract |
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-smooth muscle actin, were observed. MDCK cells expressing HOXB7 exhibited properties of migration and invasion. Unlike MDCK vector–transfected cells, MDCK-HOXB7 cells formed highly vascularized tumors in mice. MDCK-HOXB7 cells overexpressed basic fibroblast growth factor (bFGF), had more active forms of both Ras and RhoA proteins, and displayed higher levels of phosphorylation of p44 and p42 mitogen-activated protein kinase (MAPK; extracellular signal–regulated kinases 1 and 2). Effects initiated by HOXB7 were reversed by specific inhibitors of FGF receptor and the Ras-MAPK pathways. These data provide support for a function for HOXB7 in promoting tumor invasion through activation of Ras/Rho pathway by up-regulating bFGF, a known transcriptional target of HOXB7. Reversal of these effects by HOXB7-specific siRNA further suggested that these effects were mediated by HOXB7. Thus, HOXB7 overexpression caused EMT in epithelial cells, accompanied by acquisition of aggressive properties of tumorigenicity, migration, and invasion. (Cancer Res 2006; 66(19): 9527-34) |
Introduction |
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Epithelial-mesenchymal transition (EMT), initially recognized as an essential step for embryogenesis in the early 1980s (9), is now considered a major mechanism for the conversion of early-stage tumors to invasive malignancies (4, 10–12). During passage through EMT, epithelial cells lose epithelial adherens and tight junction proteins, consequently lose polarity and cell-cell contacts, and undergo a dramatic remodeling of the cytoskeleton to facilitate cell motility and invasion (13). Transcriptional factors like Snail (10) and Twist (14, 15) were unveiled as key regulators in induction of EMT in breast cancer and other cancers and act by suppressing the expression of epithelial specific adhesion molecule, E-cadherin. E-cadherin expression is irreversibly lost in invasive lobular breast cancer (16). Besides these transcriptional factors, growth factors like hepatocyte growth factor (HGF; ref. 17), transforming growth factor (TGF)-ß (18), and epidermal growth factor (EGF; ref. 19), as well as matrix metalloproteinase (MMP)-3 (20), also induce EMT in various cell lines. These studies also suggested that Snail was located at the hub of these growth factor signaling pathways leading to EMT because the activated receptor tyrosine kinases could up-regulate the expression of Snail by activating the Ras-mitogen-activated protein kinase (MAPK) pathway (21). In this study, we identified HOXB7 as one of the promising candidate genes, which was overexpressed at increasingly higher levels from normal epithelial cells to primary metastatic breast tumors to bone metastatic lesions by microarray analysis of purified epithelial cells. HOXB7 was reported to be involved in tissue remodeling of the normal mammary gland (22) and was associated with the development of breast cancer (23, 24). cDNA-based comparative genomic hybridization revealed that HOXB7 was located in a novel amplicon at 17q21.3, and this amplification correlated with poor prognosis in a panel of 186 breast cancer cases (25). Overexpression of HOXB7 in SKBR3 breast cancer cells was found to directly or indirectly regulate the expression of many angiogenic and growth factors, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin 8, Ang1, Ang2, and MMP9, and resulted in the formation of well-vascularized tumors when grown as xenografts in nude mice (23, 24). In this article, we provide evidence that HOXB7 is overexpressed in primary breast carcinomas and distant metastasis to various organs. In cell culture models, we show the ability of HOXB7 to confer the biological and molecular characteristics of EMT to epithelial cells.
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Materials and Methods |
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10 ng total RNA) from each sample was amplified with the RiboAmp RNA Amplification kit (Arcturus Engineering). The amplified RNA was labeled with the ENZO BioArray HighYield RNA transcript labeling kit (Affymetrix, Santa Clara, CA). To obtain appropriate concentrations for hybridization, three bone metastasis samples were pooled. Biotin-labeled RNA samples (12 µg RNA of normal, invasive ductal carcinoma, and pooled bone metastases) were then fragmented and hybridized to the GeneChip Human Genome U133A 2.0 Array (Affymetrix). Reverse transcription-PCR, quantitative PCR, and statistical analysis. Immortalized cell lines derived from normal human mammary epithelial cells, MCF10A, normal Madin-Darby canine kidney (MDCK) breast cancer cell lines (American Type Culture Collection, Manassas, VA), normal organoid, and tumor RNA were extracted by Trizol method, and all cDNAs were prepared with 1 µg of RNA in SuperScript II (Invitrogen, Carlsbad, CA) reactions according to the instructions of the manufacturer. Reverse transcription-PCR (RT-PCR) amplifications of HOXB7, bFGF, and internal control gene 36B4 were done with the following primer pairs: HOXB7, forward 5'-AGAGTAACTTCCGGATCTA-3' and reverse 5'-TCGGCTTCAGCCCTGTCTT-3'; bFGF, forward 5'-TCAAAGGAGTGTGTGCTAACCG-3' and reverse 5'-CTGCCCAGTTCGTTTCAGTG-3'; and 36B4, forward 5'-GATTGGCTACCCAACTGTTGCA-3' and reverse 5'-CAGGGGCAGCAGCCACAAAGGC-3'.
Quantitative real-time PCR was done and analyzed essentially as described (12) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for normalization. Primer sequences of HOXB7 for Q-PCR were as follows: forward 5'-AGAGTAACTTCCGGATCTA-3' and reverse 5'-CAGGTAGCGATTGTAGTG-3' with the TaqMan probe FAM-ACCCCTGGATGCGAAGCTCA-TAMRA (Applied Biosystems, Foster City, CA). Two-tailed Student's t test was used for analysis of the real-time PCR results.
Plasmids, siRNA, and transfection. The expression vector for Flag-tagged HOXB7 was a kind gift from Dr. Judith Gasson (University of California at Los Angeles, Los Angeles, CA). Wild-type HOXB7 was amplified with primers B7-F (5'-ATGAGTTCATTGTATTATGCGAATG-3') and B7-R (5'-ACTCTTCCTCTTCCTCCTCTGCTTCAG-3') and subcloned into pcDNA3.1-V5-His-Topo vectors (Invitrogen). Flag-tagged HOXB7 and wild-type HOXB7 plasmids were transfected into MCF10A or MDCK cells with Genejammer (Stratagene, La Jolla, CA) and cells were selected in 800 µg/mL G418–containing medium to establish stable clones. Two pairs of HOXB7 siRNA oligos [SA1, CAUUCUGUGUGUAUCUAAA (sense) and UUUAGAUACACACAGAAUG (antisense); and SA2, GGACUCUCCUUCUGUAAUA (sense) and UAUUACAGAAUUAGAGUCC (antisense)] were synthesized and transfected into MDA-MB-435 cells with Lipofectamine 2000 (Invitrogen).
Western blot, immunofluorescence assays, and antibodies. Western blot and immunofluorescence were done as described (13). Antibodies to Flag (Stratagene), HOXB7, claudin 1, claudin 4, claudin 7 (Invitrogen), E-cadherin, vimentin, ß-actin (BD Biosciences, Franklin Lakes, NJ), -smooth muscle actin (Sigma, St. Louis, MO), Ras (Santa Cruz Biotechnology, Santa Cruz, CA), MAPK, and phospho-MAPK (Cell Signaling, Danvers, MA) were used. Alexa Fluor 488–labeled phalloidin (Invitrogen) was used to detect F-actin.
Wound healing assay. For the wound healing assay, cell cultures at varying confluence were serum starved for 8 hours. After scratching the monolayer, cells were washed with PBS, cultured in 10% fetal bovine serum (FBS)-DMEM, and photographed under 10x objective lens every 3 hours.
Matrigel invasion assay. Into the Biocoat Matrigel invasion chambers (BD Biosciences) were seeded 4 x 104 cells in 2% FBS medium, and in the lower wells 10% FBS was added as the chemoattractant. After 36 hours of incubation, the filters were stained with crystal violet, and the number of cells that had penetrated through the filter was counted under x20 magnification (10 randomly selected high-power fields). For inhibition of invasion assays, the FGF receptor inhibitor Su5402 (40 µmol/L), the MAPK/extracellular signal–regulated kinase kinase (MEK) inhibitor U0126 (10 µmol/L), and the Raf inhibitor Bay43-9006 (4 µmol/L; Calbiochem, San Diego, CA) were added to the medium in the top chamber at the final concentrations indicated.
Ras and RhoA activation assay. Cell lysates were incubated with glutathione S-transferase (GST)-Raf-1 Ras binding domain or GST-Rhotekin RhoA binding domain agarose beads (Upstate, Charlottesville, VA). GTP-bound Ras or RhoA that precipitated with the beads were detected by SDS-PAGE and immunoblotting with anti-Ras antibody (Santa Cruz Biotechnology) or anti-RhoA antibody (Upstate).
Xenograft analysis. A suspension of 1 x 106 MDCK-vec or MDCK-B7 cells in 100 µL of Matrigel (BD Biosciences) was injected into the mammary fat pad (one on either side) of 10 female Swiss nu/nu mice. Tumors were removed 8 weeks later and subjected to H&E and immunohistochemical staining for histopathologic examination.
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Results |
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HOXB7 was reported to be present in a novel amplicon on chromosome 17; further fluorescence in situ hybridization (FISH) analysis of 346 tumors showed gene amplification in 10.2% of primary breast cancers, which correlated to poor prognosis (25). We determined if the overexpression of HOXB7 seen in primary tumors can be traced to gene amplification by the use of real-time PCR on tumor DNA for HOXB7 and the internal control gene GAPDH. Our analysis of DNA from the same tumor panel showed that <10% tumors had more than two copies of HOXB7 (data not shown). Thus, gene amplification seems to be the underlying mechanism accounting for only a small percentage of tumors that overexpress HOXB7 mRNA.
HOXB7, a novel factor that induces EMT. To determine whether HOXB7 protein expression plays a contributory role in tumor progression, we stably transfected FLAG-tagged HOXB7 (FB7) in MCF10A cells, an immortalized normal mammary epithelial cell line with undetectable HOXB7 expression. Intriguingly, both pooled clones and 70% (16 of 23) of the G418-selected stable clones of MCF10A-FB7 appeared spindle shaped and fibroblastic in monolayer culture, whereas HOXB7-vector control cells, like MCF10A parental cells, maintained their cobblestone-like phenotype (Fig. 2A ). This morphologic change implied that the MCF10A-FB7 cells have undergone trans-differentiation from epithelial cells to mesenchymal cells. Consistent with this observation, rearrangement of cytoskeleton as a signature of the transition was observed by phalloidin staining. In contrast with control cells exhibiting a peripheral F-actin staining with slim central stress fibers, MCF10A-FB7 cells showed a decrease in marginal F-actin but contained much thicker central stress fibers (Fig. 3B-1 ).
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The accepted paradigm of EMT dictates that cells lose markers typical of epithelial cells, such as adhesion molecules (E-cadherin) at the adherens junctions and tight junction proteins (claudins and ZO-1) at the apical junctions (13). Numerous lines of evidence have shown that loss of these proteins impairs cell-cell adhesion and cell-cell communication and facilitates dissemination of metastatic cells (28, 29). Changes in expression of these proteins were examined by Western blot and/or by immunofluorescence analyses. By Western blot analysis, expression of tight junction proteins, claudin 1 and claudin 7, was undetectable in MCF10A-FB7 pooled clones and single clones, and significantly decreased in MDCK-B7 cells (Fig. 3A). Curiously, unlike MCF10A-FB7 cells, expression levels of E-cadherin and claudin 4 were not dramatically changed in MDCK-B7 cells (Fig. 3A). However, by immunofluorescence staining, in contrast to the MDCK control cells where both E-cadherin and claudin-4 proteins had clear membrane peripheral staining pattern, in MDCK-B7 cells their distributions were mainly diffusely cytoplasmic (Fig. 3B, 2 and 5).
During EMT, loss of epithelial markers is usually accompanied by the expression of markers typical of mesenchymal cells. As shown in Fig. 3B, de novo -smooth muscle actin expression was observed in MCF10A-FB7 cells, and vimentin expression level was dramatically higher in MDCK-B7 cells (Fig. 3A). In MDCK control and pooled MDCK-HOXB7 cells, although fluorescence signals were observed in both control and HOXB7 transfectants, their expression patterns were completely different. In MDCK control cells, both -smooth muscle actin and vimentin were localized in a concentrated and polarized pattern. However, in MDCK-B7 cells, -smooth muscle actin was mainly distributed along the lamellipodia and a network of vimentin intermediate filaments was also clearly visible (Fig. 3B, 6 and 7). Thus, changes in morphology and molecular markers in both MCF10A and MDCK cells stably expressing HOXB7 were consistent with EMT.
HOXB7 can promote migration and invasion. The essential contribution of EMT to carcinoma progression is that dissociated epithelial cells acquire migration and invasive ability and are able to actively pass through the basement membrane and traverse to distant organs. To test whether HOXB7-overexpressing cells acquire greater migration and invasive ability, two assays were done: the wound healing and the Matrigel invasion assays. The wound healing assay was conducted at different confluence levels of both MDCK-vec and pooled MDCK-B7 cells. Figure 4A shows representative photomicrographs taken 0, 6, 9, and 15 hours after the cell surface was scratched for the wound healing assay. No motility was observed in MDCK-vec cells during the entire observation period, whereas the pooled MDCK-B7 cells started to fill the wound as early as 6 hours after scratching. Pooled MDCK-B7 cells also showed a significantly greater invasive potential than MDCK-vec cells in the Matrigel invasion assay (Fig. 4B and C). Moreover, lamellipodia-like structures, an important signature of cell migration, were observed in the majority of pooled MDCK-B7 cells that penetrated and traversed the Matrigel (Fig. 4B).
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, and FGF-2, have been shown to assist EMT by binding to their cognate receptors on epithelial cells and by initiating signal transduction cascades (13). The Ras-Raf-MAPK pathway has been shown to be an indispensable link in the chain of signal transduction leading to induction of EMT (18, 30). To test the involvement of this pathway in EMT induced by HOXB7 in MDCK cells, we employed the GST pull-down assay to analyze the active forms of both RhoA and Ras. In contrast to MDCK-vec cells, pooled clones of MDCK-B7 cells had more active forms of both Ras and RhoA proteins (Fig. 5A). Investigating a role for MAPK activation by testing for p44/p42 MAPK, higher levels of activation of MAPK were observed in pooled MDCK-B7 cells compared with the MDCK-vec cells, with no change in the amount of total protein (Fig. 5A). That this activation is attributable to HOXB7 was tested by suppressing endogenous HOXB7 expression in MDA-MB-435 cells using HOXB7 siRNA. Suppression of HOXB7 expression abrogated activation of the Ras-MAPK pathway (Fig. 5C), suggesting a key role for HOXB7 in this phenomenon. Further confirmation was sought that activation of these pathways is associated with the invasive ability of MDCK-B7 cells. MDCK-vec and pooled MDCK-B7 cells were seeded into Matrigel invasion chambers and treated with the RAF inhibitor Bay43-9006 or the MEK inhibitor U0126. Treatment of the cells with either inhibitor alone or in combination resulted in the complete suppression of the invasive phenotype displayed by the MDCK-B7 cells (Fig. 5B). Similar results were observed in migration assays (data not shown).
bFGF, a wide-spectrum factor functioning in mitogenesis (31), angiogenesis (32), and neurogenesis (33), was previously reported as a directly regulated gene target of HOXB7 (34). bFGF has also been shown to induce EMT in lens cells (35) in response to injury and in kidney cells (36). Furthermore, activation of FGF receptors by autocrine bFGF results in the recruitment and phosphorylation of adaptor protein SHC, which then creates binding sites for the growth factor receptor binding protein-2 adaptor in complex with the Ras-activating nucleotide exchange factor SOS (37). To verify whether this signal transduction pathway is involved in the HOXB7-induced invasive properties in our system, we first tested for bFGF expression by RT-PCR in both pooled MDCK-B7 cells and MDCK-B7 xenograft tumors grown in nude mice. Figure 6B
shows that bFGF was expressed at higher levels in both MDCK-B7 cells and transplanted tumors compared with MDCK-vector control cells. Treatment of MDCK-B7 cells with the FGF receptor–specific inhibitor Su5402 (Calbiochem) could attenuate the Ras-GTP form (Fig. 6D). Further, the FGF receptor–specific inhibitor could inhibit invasion ability of MDCK-B7 cell by 80% (Fig. 6D). The expression of some other well-known EMT regulators, such as members of the TGF-ß pathway and members of the Snail family, was also determined by RT-PCR or Western blot analysis, but no significant change was detected (data not shown). Thus, several lines of evidence point to bFGF as the major mediator of EMT initiated by HOXB7.
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Discussion |
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These morphologic and cell-cell contact changes ultimately reflect on cell mobility and invasive ability. It is well known that small GTP binding proteins, such as members of the Ras and Rho families, comprehensively regulate cell migration and invasion. Also well documented is that the Ras-RAF-MAPK pathway plays indispensable role in EMT induced by activation of receptor tyrosine kinase of growth factors like HGF, VEGF, EGF, and bFGF (30). Taking into account published findings that HOXB7 could directly transactivate the expression of bFGF in both melanoma and breast cancer cell lines (23, 34), we investigated bFGF expression and found that it was high in both HOXB7 stably transfected MDCK cells and the xenograft tumors. Further, blocking FGF autocrine signaling cascade with the FGF receptor inhibitor Su5402 could attenuate activation of the Ras-RAF-MAPK pathway and the invasive ability of MDCK-B7 cells. In addition to activation of the Ras pathway, more RhoA-GTP form proteins were observed in HOXB7-transfected cells. RhoA protein is known by its ability to remodel the actin cytoskeleton and form thick stress fibers, which are required for migratory behavior of cells (40). Using MDCK cells, Zondag et al. (27) have shown that a shift in balance between RhoA and RAC activity can control the transition of phenotype from epithelial to mesenchymal. They found that sustained signaling by oncogenic RasV12 permanently down-regulated RAC activity, which led to up-regulation of RhoA activity and EMT. On the other hand, reconstitution of RAC activity by expression of Tiam1 or RACV12 led to down-regulation of Rho activity and restored an epithelial phenotype to mesenchymal, RasV12-transformed cells (27). Although activation of RhoA mediates formation of F-actin stress fibers and enhances cell motility, some studies indicate that excessive activation of RhoA can actually inhibit polarization and motility (41, 42). For example, stimulation of U118 cells with S1P resulted in a 5-fold induction of RhoA activity and inhibition of migration (42). In the case of HOXB7-transformed MDCK cells, higher bFGF expression was observed compared with vector controls (Fig. 6B). The expression of bFGF could result in constitutively activating Ras signaling through autocrine signaling cascades. Activated Ras signaling pathways could further activate RhoA by 2-fold (Fig. 5A), which could have mediated the formation of contractile stress fiber to facilitate migration and invasion.
This study was initiated from the finding that HOXB7 is an mRNA that showed a stepwise increase in expression during breast cancer progression from primary tumor to distant metastasis. But what is the cause of the abnormally high expression of HOXB7 in breast carcinomas? Kallioniemi's group first reported that overexpression of HOXB7 was related to breast cancer by identification of HOXB7 in a novel amplicon, and that amplification of the gene in 10.2% of primary breast cancers correlated with poor prognosis (25). Our data provide evidence that may mechanistically link the high expression of HOXB7 to poor prognosis of breast cancer.
In our studies, the percentage of tumors displaying HOXB7 expression in comparison with normal tissues was much higher (60% at 5-fold higher than normal) compared with the incidence of gene amplification (10.2%) revealed by FISH analysis (25). Our assessment of HOXB7 gene copy number by real-time PCR in the same tumor panel showed that <10% tumors had more than two copies of HOXB7 (data not shown). This implied that not just genomic amplification but also other transcriptional regulation mechanisms are involved in overexpression of HOXB7 in breast carcinomas. Although homeobox genes have been known for several decades, the mechanism by which their expression is regulated remains elusive. Periodic expression pattern of HOXB7 during lactation cycles in mice suggests that HOXB7 expression may be under the control of hormones like estrogen, progesterone, and prolactin. In addition, HOXB7 expression could also be controlled by extracellular matrix (22).
Taken together, we show that HOXB7 was highly expressed in both primary metastatic tumors and bone metastasis, and overexpression HOXB7 could induce EMT, which is associated with tumor progression and poor prognosis. Further studies to identify mechanisms regulating HOXB7 expression and HOXB7 downstream regulated genes would be worthwhile to better understand the pathogenic role of HOXB7 in tumorigenesis and explore the potential of HOXB7 as a therapeutic target.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Judith Gasson for generously providing us with the HOXB7 expression plasmid; Marc Halushka, M.D., Ph.D., for metastatic breast cancer tissues through the Breast Cancer Rapid Autopsy Program funded by Department of Defense; Dr. Ingo Runnebaum, M.D., for kindly providing the FGF-R inhibitor; and Tsuyoshi Mori for help with animal experiments.
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Footnotes |
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Received 12/16/05. Revised 6/ 1/06. Accepted 7/21/06.
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