Breast Cancer Metastasis Suppressor-1 Differentially Modulates Growth Factor Signaling

Abstract

That metastatic tumor cells grow in selective non-native environments suggests an ability to differentially respond to local microenvironments. BRMS1, like other metastasis suppressors, halts ectopic growth (metastasis) without blocking orthotopic tumor formation. BRMS1-expressing tumor cells reach secondary sites but do not colonize distant tissues, compelling the hypothesis that BRMS1 selectively restricts the ability of tumor cells to respond to exogenous regulators in different tissues. Here we report that BRMS1 expression in metastatic human breast cancer cells leads to a selective reduction in epidermal growth factor receptor expression and downstream (AKT) signaling. Signaling through another receptor tyrosine kinase, hepatocyte growth factor receptor (c-Met), remains unaltered despite reduced levels of the signaling intermediate phosphatidylinositol (4,5)-bisphosphate. Interestingly, reduced downstream calcium signaling is observed following treatment with platelet-derived growth factor, consistent with decreased phosphatidylinositol (4,5)-bisphosphate. However, platelet-derived growth factor receptor expression is unaltered. Thus, BRMS1 differentially attenuates cellular responses to mitogenic signals, not only dependent upon the specific signal received, but at varying steps within the same signaling cascade. Specific modulation of signaling responses received from the microenvironment may ultimately dictate which environments are permissive/restrictive for tumor cell growth and provide insights into the biology underlying metastasis. That metastatic tumor cells grow in selective non-native environments suggests an ability to differentially respond to local microenvironments. BRMS1, like other metastasis suppressors, halts ectopic growth (metastasis) without blocking orthotopic tumor formation. BRMS1-expressing tumor cells reach secondary sites but do not colonize distant tissues, compelling the hypothesis that BRMS1 selectively restricts the ability of tumor cells to respond to exogenous regulators in different tissues. Here we report that BRMS1 expression in metastatic human breast cancer cells leads to a selective reduction in epidermal growth factor receptor expression and downstream (AKT) signaling. Signaling through another receptor tyrosine kinase, hepatocyte growth factor receptor (c-Met), remains unaltered despite reduced levels of the signaling intermediate phosphatidylinositol (4,5)-bisphosphate. Interestingly, reduced downstream calcium signaling is observed following treatment with platelet-derived growth factor, consistent with decreased phosphatidylinositol (4,5)-bisphosphate. However, platelet-derived growth factor receptor expression is unaltered. Thus, BRMS1 differentially attenuates cellular responses to mitogenic signals, not only dependent upon the specific signal received, but at varying steps within the same signaling cascade. Specific modulation of signaling responses received from the microenvironment may ultimately dictate which environments are permissive/restrictive for tumor cell growth and provide insights into the biology underlying metastasis. Metastasis begins with dissemination of cells from the primary tumor and entails survival in the bloodstream and subsequent arrival and proliferation at secondary sites (1Eccles S.A. Welch D.R. Lancet. 2007; 369: 1742-1757Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). However, following seeding at different organ sites, growth of tumor cells is nonrandom, depending on the cancer type. Organotropism of metastasis was first characterized by Sir Stephen Paget in his “seed and soil” hypothesis. Paget noted that breast cancer metastases were more frequently observed in bone and lymph nodes than at other sites in the body. Recent findings extend his observations with the concept of a “pre-metastatic niche” created by tumor cells for their eventual arrival at secondary sites (2Kaplan R.N. Riba R.D. Zacharoulis S. Bramley A.H. Vincent L. Costa C. MacDonald D.D. Jin D.K. Shido K. Kerns S.A. Zhu Z. Hicklin D. Wu Y. Port J.L. Altorki N. Port E.R. Ruggero D. Shmelkov S.V. Jensen K.K. Rafii S. Lyden D. Nature. 2005; 438: 820-827Crossref PubMed Scopus (2383) Google Scholar). To ensure survival, the tumor cell must be able to respond to microenvironmental cues at every step of the metastatic cascade, especially at secondary sites. Secondary tumor growth likely parallels growth at the primary site, the major distinction being that tumor cells find themselves in non-native ectopic environments. Interfering with a tumor cell's ability to interact with microenvironmental factors would therefore be expected to limit its capacity to proliferate. Importantly, arrival and seeding at particular sites do not guarantee growth. Metastasis suppressors are a growing family of more than 20 proteins that, when re-expressed in metastatic cells, block metastasis without preventing tumor growth at orthotopic sites (3Vaidya K.S. Welch D.R. J. Mamm. Gland Biol. Neopl. 2007; 12: 175-190Crossref PubMed Scopus (34) Google Scholar). Their mechanisms of action have not been fully elucidated, but many appear to block growth at secondary sites by interfering with the tumor cell's ability to respond to its microenvironment (1Eccles S.A. Welch D.R. Lancet. 2007; 369: 1742-1757Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). BRMS1 (breast cancer metastasis suppressor-1)-expressing tumor cells maintain the ability to form primary tumors and, despite maintaining the ability to seed secondary sites, do not colonize distant tissues (4Phadke P.A. Vaidya K.S. Nash K.T. Hurst D.R. Welch D.R. Am. J. Pathol. 2008; 172: 809-817Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Because BRMS1 selectively determines where cells grow or not, our objective in this study was to begin testing the hypothesis that BRMS1 differentially regulates growth factor responses. Cell Lines and Cell Culture—MDA-MB-231 and MDA-MB-435 human breast carcinoma cells were transfected with a lentiviral vector construct expressing BRMS1 under the control of a cytomegalovirus promoter (4Phadke P.A. Vaidya K.S. Nash K.T. Hurst D.R. Welch D.R. Am. J. Pathol. 2008; 172: 809-817Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The origin of MDA-MB-435 cells has recently been called into question. However, MDA-MB-435 cells have been demonstrated to express milk proteins and metastasize from mammary fat pad but not subcutaneous tissue, consistent with the cell line being a breast carcinoma (5Sellappan S. Grijalva R. Zhou X.Y. Yang W.T. BarEli M. Mills G.B. Yu D.H. Cancer Res. 2004; 64: 3479-3485Crossref PubMed Scopus (136) Google Scholar, 6Welch D.R. Clin. Exp. Metastasis. 1997; 15: 272-306Crossref PubMed Scopus (162) Google Scholar). Three single-cell clones were chosen from each cell line: 231BRMS1.9, 231BRMS1.10, 231BRMS1.12, 435BRMS1.10, 435BRMS1.13, and 435BRMS1.14. MDA-MB-231/435 vector transfectants (231/435) and 231BRMS1/435BRMS1 clones were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM 6The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; HGF, hepatocyte growth factor; PtdIns(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; shRNA, small hairpin RNA. /F-12) supplemented with 1% nonessential amino acids and glutamine (Invitrogen) and containing 5% fetal bovine serum. Parental 231 and 231BRMS1 cells were passaged using 0.125% trypsin and 2 mm EDTA (Invitrogen) in Ca2+/Mg2+-free phosphate-buffered saline, and parental 435 and 435BRMS1 cells were passaged using 2 mm EDTA. Cell lines were confirmed to be free of Mycoplasma contamination. No antibiotics or antimycotics were used during routine culture. Wherever growth factor treatment was required, cells were cultured under serum-free conditions (DMEM/F-12) for 24 h prior to treatment. Immunoblotting and Immunoprecipitation—Cells were rinsed twice with ice-cold phosphate-buffered saline and lysed in a buffer containing 25 mm Tris-HCl (pH 7.4), 50 mm β-glycerol phosphate, 0.5 mm EDTA, 5% glycerol, 0.1% Triton X-100, 1 mm sodium orthovanadate, 1 mm benzamidine, and protease inhibitor mixture containing aprotinin, leupeptin, and phenylmethylsulfonyl fluoride (Roche Applied Science). Protein concentration was determined using a Bradford colorimetric assay (Pierce). Protein was denatured with Laemmli buffer at 95 °C for 5 min, and lysate equal to 50 μg was loaded into each well. Proteins were separated using either 8 or 12% SDS-polyacrylamide gels, and resolved proteins were transferred to polyvinylidene difluoride membranes. Membranes were incubated in Tris-buffered saline containing 0.05% Tween 20 and 5% fat-free dry milk for 1 h at room temperature. Membranes were incubated with primary antibodies to epidermal growth factor receptor (EGFR; 2232), p42/p44 mitogen-activated protein kinase (MAPK; 9102), or phospho-p42/p44 MAPK (9101) (Cell Signaling, Danvers, MA) or phospho-AKT-Ser473 (P4112) or AKT (P2482) (Sigma) overnight at 4 °C and subsequently with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Signals were visualized using ECL (Amersham Biosciences) following the manufacturer's instructions. For immunoprecipitation, cell lysate (500 μg) was incubated with 1 μg of primary antibody for 1 h, followed by incubation with 15 μl of Protein A/G beads (IP05, Calbiochem) for 1 h at 4 °C in a total volume of 500 μl with cell lysis buffer. Beads were pelleted at 13,000 × g for 1 min and washed with buffer containing 300 mm NaCl, 50 mm Tris-HCl for a total of three washes. Protein was denatured by addition of 25 μl of Laemmli buffer to the bead pellet and boiling at 95 °C for 5 min. Immunoprecipitated proteins in the supernatant were resolved by SDS-PAGE, followed by immunoblotting as described above. shRNA for BRMS1—435 or 435BRMS1 cells were transfected with a shRNA oligonucleotide against BRMS1 or a nonspecific control shRNA (V2HS_72900, Open Biosystems) using RNA-intro (RHS3600, Open Biosystems) according to the manufacturer's instructions. Medium was replaced 6 h following transfection with complete DMEM. Following transfection, cells were harvested for RNA with TRIzol (Invitrogen) reagent after 5 days, and mRNA levels were determined by real-time quantitative PCR. Taqman Quantitative Real-time PCR—mRNA levels of BRMS1, EGFR, and endogenous ribosomal S9, as an internal reference (7Blanquicett C. Johnson M.R. Heslin M. Diasio R.B. Anal. Biochem. 2002; 303: 209-214Crossref PubMed Scopus (99) Google Scholar), were quantified using real-time PCR analysis (Taqman) on an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA). Total cellular RNA was extracted using TRIzol, and ethanol-precipitated RNA was resuspended in 60 μl of diethyl pyrocarbonate-treated water. Amplification of specific PCR products was detected using fluorescent probes labeled with 6-carboxyfluorescein at the 5′ end. PCR was performed in a total reaction volume of 50 μl containing 4 mm MgCl2, 200 μm each dNTP, 1.25 units of Taq polymerase, and 3 μl of total RNA. The relative mRNA expression of the target genes was quantified using the comparative cycle time method. Each sample was assayed in triplicate. Immunocytochemistry—Cells were plated on glass coverslips and serum-starved for 24 h. Cells were subsequently treated with and without EGF (50 ng/ml) for 30–60 min, fixed using 4% paraformaldehyde for 20 min, and permeabilized using 0.5% Triton X-100 for 8 min. F-actin was stained using rhodamine-conjugated phalloidin; nuclei were counter-stained using 4′,6-diamidino-2-phenylindole, and cells were viewed under a Olympus IX-70 inverted epifluorescence microscope. Representative images were obtained, combined, and processed using IPLab (v3.7) deconvolution software (Scanalytics, Rockville, MD) at the University of Alabama at Birmingham High Resolution Imaging Core facility. Ratiometric Calcium Imaging—Cells grown on coverslips were incubated in medium containing 5 μm Fura-2/AM (Molecular Probes) from a 5 mm dimethyl sulfoxide stock for 60 min. After incubation, coverslips were washed twice and transferred to a glass plate with an 8-mm hole, which was sealed with vacuum grease, forming a well to which 50 μl of medium was added. Cells were illuminated with a Lambda DG-4 light, and images were collected with a Coolsnap HQ camera controlled by Metafluor imaging software (Molecular Devices, Sunnyvale, CA). Multiple cells within the field of view were tracked as individual data sets; images were collected (one image/s), and ratio values were saved. Excitation wavelengths were 340 and 380 nm, and data were collected at 510 nm; the ratio was a result of the 340/380 image. For collection of ratiometric data from growth factor-stimulated cells, the experiments were carried out as above, except cells grown on coverslips were incubated in serum-free medium for 3 h prior to loading with Fura-2/AM. Ratiometric data were collected for 6–10 min to confirm a stable base line. EGF was then added to the medium, and data collection was continued. Ionophore A23187 (Calbiochem) was added at the end of the experiment to demonstrate that all cells still mobilized calcium. For inhibitor studies in EGF-stimulated cells, the cells were treated with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 μm, Calbiochem) for 45 min prior to EGF stimulation. Immunohistochemistry—BRMS1 expression was assessed using a monoclonal antibody (3a1.21) as described previously S. S. N. S. M. D. N. Welch D.R. Clin. Cancer Res. 12: PubMed Scopus Google Scholar). were glass and antibody and detection was performed using a peroxidase-conjugated secondary was using antibody at a of using to the cells were plated in and to for 24 h. of of Johnson J. Google Scholar), or were transfected in cells in to a total concentration of 2 using reagent (Invitrogen) in Medium was replaced with medium after 6 h. Following transfection, cells were lysed after h, and were at °C and used the was using using a were to and data were as relative where control was at 435BRMS1 cells were in at a seeding of and to for 24 h. Cells were transfected with μg) or in with BRMS1 small interfering RNA or control small interfering RNA with μg) was used as a transfection interfering were transfected at a concentration of 5 Medium was replaced 24 h and cell were collected and at was as described above. BRMS1 and have previously that BRMS1-expressing cells grow in orthotopic sites, but not ectopic sites Welch D.R. Cancer Res. Google Scholar, Welch D.R. Clin. Exp. Metastasis. Scopus (99) Google Scholar, D.R. Y. Vaidya K.S. R. Welch D.R. J. Biol. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). of BRMS1 to in (4Phadke P.A. Vaidya K.S. Nash K.T. Hurst D.R. Welch D.R. Am. J. Pathol. 2008; 172: 809-817Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Metastasis then be by BRMS1-expressing cells secondary sites. tumor cells still ectopic sites, but to and that survival in non-native environments during and at ectopic tumor cells to interact with and respond to their through and downstream survival receptor expression of EGFR, platelet-derived growth factor receptor and hepatocyte growth factor receptor following BRMS1 receptor tyrosine are to individual in the metastatic cascade, and expression was by immunoblotting in BRMS1-expressing clones from 231 and 435 cells and with protein expression was reduced in 231BRMS1 cells, protein was in 435BRMS1 in protein expression in BRMS1-expressing was not detected in either cells or their BRMS1-expressing not of the of to be in 231BRMS1 and 435BRMS1 cells with mRNA expression was by real-time quantitative PCR in BRMS1-expressing clones in 231 and 435 mRNA decreased in BRMS1-expressing cells, following a to that of protein expression shRNA to BRMS1 in 435BRMS1 cells the of BRMS1 in expression Because BRMS1 is a of that J.L. Welch D.R. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), we BRMS1 expression promoter of BRMS1 with the construct for the promoter to to a Johnson J. Google in cells reduced promoter by more than shRNA BRMS1 in 435BRMS1 cells promoter in a assay To data from our cell line we performed analysis of primary human breast for and and BRMS1 were BRMS1 was to the was to the cell of BRMS1 of BRMS1 leads to a selective of the phosphatidylinositol J. D. N. Y. Welch D.R. Cancer Res. 2005; Google Scholar). 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Res. 15: PubMed Scopus (99) Google Scholar), was not that EGF or treatment to MAPK Immunoblotting to have to in AKT or MAPK following and data for treatment are not To BRMS1 the ability of to after EGF was from cell of cells, and receptor was determined after EGF treatment (50 15 tyrosine was in the 231BRMS1 cells, the of was to the reduced levels and To downstream was in to Cells treated with and without EGF (50 ng/ml) were stained with rhodamine-conjugated vector transfectants to EGF time with 231BRMS1 cells reduced and were in 435BRMS1 cells BRMS1 of of the major calcium of signaling. to and levels of in to growth factor stimulation. as a for which calcium Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, 2002; Google Scholar, Biochem. PubMed Scopus Google Scholar). of calcium from is in modulation of the for and during and J. Cell PubMed Scopus Google Scholar, M. K. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). have previously demonstrated that of BRMS1 leads to a selective reduction in levels of the and levels J. D. N. Y. Welch D.R. Cancer Res. 2005; Google Scholar). EGF and treatment (50 ng/ml) calcium in parental breast carcinoma cells that was when BRMS1 was re-expressed that (50 ng/ml) treatment of 435BRMS1 cells to a calcium J. D. N. Y. Welch D.R. Cancer Res. 2005; Google was consistent with the of in in calcium was observed following treatment not imaging and are being and are of with breast cancer metastases to following primary tumor C. Am. J. Pathol. PubMed Scopus Google Scholar, R. J. J. 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PubMed Scopus Google Scholar), and BRMS1-expressing cells reach secondary sites with the parental of the is in survival 2007; PubMed Scopus Google Scholar, M. Cell PubMed Scopus Google Scholar). data with decreased of downstream of the of in BRMS1-expressing cells (4Phadke P.A. Vaidya K.S. Nash K.T. Hurst D.R. Welch D.R. Am. J. Pathol. 2008; 172: 809-817Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). BRMS1-expressing cells the secondary do not form metastases (4Phadke P.A. Vaidya K.S. Nash K.T. Hurst D.R. Welch D.R. Am. J. Pathol. 2008; 172: 809-817Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). human BRMS1 expression has been with growth factor receptor expression S. S. N. S. M. D. N. Welch D.R. Clin. Cancer Res. 12: PubMed Scopus Google Scholar). observations to the hypothesis that BRMS1 expression regulates selective growth factor data the that selective of growth factor signaling may where BRMS1-expressing cells do or do not of a tumor cell with its microenvironment as well as and are for eventual growth and proliferation 2004; PubMed Scopus Google Scholar). this we have on the modulation of signaling by not an responses to three growth factors and HGF, all of which have been in breast tumor and metastatic were D. K. R. S. 2005; Google Scholar, Y. K. J. M. Cancer 2007; PubMed Scopus Google Scholar). The signaling to EGF in BRMS1-expressing cells be in to reduction or complete of decreased calcium through may be a of reduced The of not the of AKT in BRMS1-expressing cells following treatment. of AKT are M. S. M. Cell 2005; 12: PubMed Scopus Google Scholar), and through other be out and is the of this consistent with the of in in calcium was observed following treatment not by and have demonstrated decreased is downstream of in the of BRMS1 Y. D.R. Biol. PubMed Scopus Google Scholar, M. R. Welch D.R. N. Cancer Res. 2005; PubMed Scopus Google Scholar). of downstream is expected with different growth is that BRMS1 signaling through the same at different levels of the signaling cascade. different signaling of the to be more to than the MAPK cascade. The of BRMS1 remains BRMS1 may either expression or factors or another signaling intermediate that determines as of the BRMS1 However, the of a BRMS1 antibody testing this hypothesis. have recently that of with of BRMS1 expression with of the first of BRMS1 expression is still in all BRMS1 BRMS1 still maintain the ability to that selective may be a of the of the BRMS1 D.R. Y. Vaidya K.S. R. Welch D.R. J. Biol. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). of EGFR, but not or the of BRMS1 the in selective we and have previously specific with BRMS1 However, the target of BRMS1 and the primary to be experiments are in their of the of microenvironments. experiments do not the of different factors on the tumor cell in the relative expression and the of growth factors in the individual microenvironment are not and expression of growth factor in a particular tissue, is not that cancer cells colonize a as demonstrated in our is to individual growth factors in by analysis of receptor levels with signaling responses in tumor BRMS1-expressing cells do not major in in that different steps in the metastatic Welch D.R. Clin. Exp. Metastasis. Scopus (99) Google Scholar), our of in to end the and that signaling our data that a that or levels within the same be for the treatment of our data begin to the for metastasis we fully the for growth at the orthotopic ectopic sites, in in addition to the tumor cell may be major in signaling within the tumor cell demonstrated in our may a that factor which determines where tumor cells are to Johnson of for the of the Welch for and with