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[Cancer Research 65, 713-717, February 1, 2005]
© 2005 American Association for Cancer Research

Priority Reports

Metastasis Suppression by Breast Cancer Metastasis Suppressor 1 Involves Reduction of Phosphoinositide Signaling in MDA-MB-435 Breast Carcinoma Cells

Daryll B. DeWald1,2, Javad Torabinejad1,2, Rajeev S. Samant4, Derrick Johnston1,2, Nuray Erin3, Joseph C. Shope1, Yi Xie4 and Danny R. Welch2,4

1 Department of Biology, Utah State University, Logan, Utah; 2 National Foundation for Cancer Research, Center for Metastasis Research; 3 Jake Gittlen Research Institute, The Pennsylvania University College of Medicine, Hershey, Pennsylvania; and 4 Department of Pathology, Comprehensive Cancer Center, The University of Alabama at Birmingham, Birmingham, Alabama

Requests for reprints: Danny R. Welch, Department of Pathology, University of Alabama at Birmingham, 1670 University Drive, Volker Hall, Room G019A, Birmingham, AL 35294-0019. Phone: 205-934-2956; Fax: 205-975-1126; E-mail: dwelch{at}path.uab.edu or Daryll B. DeWald, 5305 University Blvd., Logan, UT 84322-5305. Phone: 435-797-3711; E-mail: dewald{at}biology.usu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Several molecules that suppress metastasis without suppressing tumorigenicity have been identified, but their mechanisms of action have not yet been determined. Many block growth at the secondary site, suggesting involvement in how cells respond to signals from the extracellular milieu. Breast cancer metastasis suppressor 1 (BRMS1)–transfected MDA-MB-435 cells were examined for modifications of phosphoinositide signaling as a potential mechanism for metastasis suppression. 435/BRMS1 cells expressed <10% of phosphatidylinositol-4, 5-bisphosphate compared with parental cells, whereas levels of the PtdIns(4)P and phosphatidylinositol-3-phosphate were unchanged. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] were decreased in 435/BRMS1 cells by ~50%. Phosphatidylinositol-3,4,5-trisphosphate levels were undetectable in 435/BRMS1 cells, even when stimulated by exogenous insulin or platelet-derived growth factor. Immunofluorescence microscopy to examine cellular distribution confirmed that phosphatidylinositol-4,5-bisphosphate distribution with cells was unchanged but was uniformly decreased throughout the cell. Although the gross morphology of 435/BRMS1 cells is similar to the parent, filamentous actin was more readily apparent in 435/BRMS1. Intracellular calcium, measured using Fluo-3 and Fura-2 fluorescent calcium indicator dyes, was somewhat lower, but not statistically different in 435/BRMS1 compared with parental cell. However, when stimulated with platelet-derived growth factor, MDA-MB-435 cells, but not 435/BRMS1 cells mobilized intracellular calcium. Taken together, these results implicate signaling through phosphoinositides in the regulation of breast cancer metastasis, specifically metastasis that can be suppressed by BRMS1.

Key Words: metastasis suppressor gene • microenvironment • tumor-host interactions • cell signaling


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Metastasis suppressors are a relatively new class of proteins defined by their ability to suppress formation of secondary tumor masses without blocking growth of neoplastic cells at orthotopic or subcutaneous sites. To date, 14 metastasis suppressors have been discovered (1), but their mechanisms of action remain largely unknown. Breast cancer metastasis suppressor 1 (BRMS1) was discovered using differential display comparing mRNA expression levels between metastasis-competent MDA-MB-435 and metastasis-suppressed, human breast carcinoma cell lines that had received an intact copy of human chromosome 11 by microcell-mediated chromosome transfer. Constitutive expression of BRMS1 in human breast (MDA-MB-435 and MDA-MB-231), murine mammary (4T1 and 66cl4), and human melanoma (C8161 and MelJuSo) cells results in significant suppression of metastasis without blocking tumor formation. Rare metastases that developed had lost expression of BRMS1 (2).

BRMS1-transfected cells are equally invasive to their metastatic parents and have been found in the vasculature of mice bearing orthotopic tumors, suggesting that BRMS1, like many metastasis suppressors, affects late steps in the metastatic cascade, particularly a cell's ability to proliferate at secondary sites. BRMS1 protein localizes predominantly (>90%) to the nucleus, restores gap junctional intercellular communication and is a component of the mSin3a family of histone deacetylase complexes (3). This study was initiated in order to test the hypothesis that BRMS1 expression could regulate second messengers that, in turn, could affect growth and other cellular processes.

Because metastasis involves multiple cell-cell and cell-matrix interactions, a multitude of signaling changes have been implicated in various steps of the metastatic cascade. For the current study, the focus was on phosphoinositides because modulation of intracellular calcium, a downstream effector of phosphoinositide signaling, can affect invasion through Matrigel-coated membranes and metastasis in vivo (4). Because phosphoinositides and inositol phosphates are key regulators of cellular calcium signaling, we tested whether changes in BRMS1 expression alter phosphoinositide levels and/or localization. We found that BRMS1 expression led to a dramatic, and relatively specific, reduction of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] levels, suggesting that metastasis suppression by BRMS1 may be due to regulation of phosphoinositide signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell Lines and Cell Culture. MDA-MB-435 and MDA-MB-231 human breast carcinoma cells were cultured in a 1:1 mixture of DMEM and Ham's F12 medium supplemented with 5% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 1% nonessential amino acids, and 1 mmol sodium pyruvate. BRMS1-transfected cells also received 500 µg/mL G-418 (Geneticin; Invitrogen, San Diego, CA). Cells were cultured on 100-mm Corning tissue culture dishes at 37°C with 5% CO2 in a humidified atmosphere.

Cells were passaged at 80% to 90% confluence using a 2 mmol EDTA solution in Ca2+/Mg2+-free Dulbecco's PBS. BRMS1 was cloned into the constitutive mammalian expression vector pcDNA3 (Invitrogen) under control of the cytomegalovirus promoter. No antibiotics or antimycotics were used. All cell lines were negative for Mycoplasma sp. contamination using a PCR-based assay (TaKaRa, Madison, WI).

Radiochemicals. myo-[2-3H]inositol (10-25 Ci/mmol) was purchased from Perkin-Elmer Life Sciences (Boston, MA). For high-performance liquid chromatography standards, [32P]PtdIns(3)P and PtdIns(4)P were from in vitro phosphorylation reactions with yeast phosphatidylinositol 3-kinase or phosphatidylinositol 4-kinase and [{gamma}32P]ATP, followed by separation and extraction by thin-layer chromatography as previously described (5). PtdIns(4,5)P2 [inositol-2-3H] and Ins(1,4,5)P3 [D-inositol-1-3H] were purchased from Perkin-Elmer, and [32P]PtdIns(3,4)P2 and [32P]phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] were prepared by in vitro phosphorylation of PtdIns(4)P and PtdIns(4,5)P2, respectively, by mammalian phosphatidylinositol 3-kinase with [{gamma}-32P]ATP, followed by thin-layer chromatography purification (6).

Radiolabeling Cells and Lipid Analysis. MDA-MB-435, MDA-MB-231, 231/BRMS1 and 435/BRMS1 cells were grown to 60% to 80% confluence in 75 cm2 flasks, washed with Ca2+/Mg2+-free Dulbecco's PBS and labeled with myo-[2-3H]inositol (20 µCi/mL; 24 hours) in inositol-free DMEM containing 10% calf serum. After 24 hours, the medium was replaced. For experiments where growth factor stimulation [platelet-derived growth factor (PDGF)] was included, cells were serum-deprived for 2 hours in inositol-free and serum-free DMEM containing 0.2% bovine serum albumin and 10 µCi/mL myo-(2-3H)-inositol, followed by PDGF (50 ng/mL) stimulation and harvest. Ice-cold trichloroacetate was added to the flasks to a final concentration of 10% and incubated on ice for 1 hour before scraping and placing the liquid in 15 mL conical screw-cap centrifuge tubes. Cells were collected by centrifugation (5 minutes, 20,000 x g) before resuspension in 5 mL 5% trichloroacetate in a 1 mmol EDTA solution before recentrifugation and lipid extraction.

Lipids were extracted from the cell pellet by resuspending cells in 0.75 mL chloroform/methanol/HCl (40:80:1 v/v/v) and vortexing vigorously every 60 seconds for 15 minutes. Then, 0.25 mL of chloroform and 0.45 mL of 0.1 mol/L HCl were added to the cells and they were vortexed for 2 minutes, centrifuged (17,500 x g for 2 minutes), and the bottom, organic layer was transferred to another tube for continued processing. Ammonia (50 µL of a 1 mol/L solution) was added and the solutions in the tubes were dried.

Lipids were deacylated as described (6) with minor modifications. Dried lipids were resuspended in 0.5 mL of methylamine reagent (42.8% of 25% methylamine, 45.7% of methanol, 11.4% of n-butanol) by bath sonication, incubated at 53°C for 50 minutes, and dried under reduced pressure. Deacylated lipids were suspended in 0.75 mL H2O by sonication and extracted thrice with 0.5 mL n-butanol/petroleum ether/ethyl formate (20:4:1 v/v). The aqueous phase was dried under reduced pressure and suspended in 200 µL of H2O. An aliquot (10-20 µL) of each sample was used to determine the radioactivity by liquid scintillation counting. For preparation of loading samples for HPLC, standardization was done using the 3H counts, which approximates phosphatidylinositol content.

Phosphoinositides were resolved with a mobile phase of ammonium phosphate (pH 3.8) using strong anion exchange Partisil 10 SAX (4.6 x 250 mm) columns (Whatman, Clifton, NJ). Anion-exchange columns were fitted with guard columns (SecurityGuard; Phenomenex, Torrance, CA) containing strong anion exchange inserts. The gradients for separation of glycerophosphoinositols (gPI): gPI(3)P, gPI(4)P, gPI(3,4)P2, gPI(3,5)P2, gPI(4,5)P2, and gPI(3,4,5)P3 were 5 mL of 10 mmol, 60 mL of a linear gradient, 10 mmol to 0.8 mol/L, 2 mL of a linear gradient, 0.8 to 1 mol/L, 3 mL of 1 mol/L, respectively. Fractions (0.3 mL) were collected every 20 seconds, mixed with 2 mL of water-miscible scintillation cocktail, and counted in a liquid scintillation counter.

Immunolocalization of Phosphoinositides. Cover slips and slides were purchased from Fisher Scientific (Pittsburgh, PA). Formaldehyde was obtained from Ted Pella, Inc. (Redding, CA). Purified RC6F8 anti-PtdIns(3,4,5)P3 IgM and 2C11 anti-PtdIns4,5)P2 IgM were purchased from Echelon Biosciences, Inc. (Salt Lake City, UT). Fluorophore-tagged (Texas red) anti-mouse IgM secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Cells analyzed during the logarithmic stage on cover slips were washed with cold TBS and fixed with 2% formaldehyde, then permeabilized with 0.5% Triton X-100 in TBS. After blocking with 10% goat serum in TBS, either RC6F8 monoclonal antibody ascites (1:50 dilution) or 10F8 (1:5,000 dilution) was added and incubated for 1 hour at room temperature. After washing thrice with blocking solution, fluorophore-labeled anti-mouse IgM (1:2,000 dilution) was added and incubated at room temperature for 1 hour. After washing the cells thrice with deionized water, cells were observed using a confocal microscope (model #MRC1024; Bio-Rad, Hercules, CA).

Calcium Imaging. MDA-MB-435 or MDA-MB-435/BRMS1 cells grown on cover slips were incubated in media containing 5 µmol/L Fura-2 AM (Fura-2-acetoxymethyl ester; Molecular Probes, Eugene, OR) from a 5 mmol DMSO stock solution for 90 minutes. At the end of the incubation period, slides were washed twice and transferred to a glass plate with an 8 mm hole and sealed with vacuum grease forming a well to which 40 µL of media was added. The plate was placed on Nikon Diaphot TE200 inverted microscope using a 40x Plan Fluor lens. The cells were illuminated with a Lamda DG-4 (Sutter Instruments, Novato, CA) and images were collected with a CoolSnap HQ (Roper Scientific, Duluth, GA) camera controlled by MetaFluor (Universal Imaging, Downingtown, PA). Multiple cells within the field of viewwere tracked as a seperate data set; one image were selected per second and ratio values were saved. Excitation wavelengths were 340 and 380 nm and collected at 510nm; the ratio was the result of the 340/380 image.

For collection of ratiometric data from growth factor–stimulated cells, the experiments were carried out as above except that cells grown on cover slips were incubated in serum-free media for 2 to 4 hours prior to loading with Fura-2. PDGF (50 ng/mL) was added to the media in the well and collection of data was begun. Ratiometric data was collected for 10 minutes to confirm a stable baseline. Then PDGF was added to the media and collection of data was continued. Calcium ionophore (A23187, Calbiochem, San Diego, CA) was added at the end of the experiment to show that all cells were still able to mobilize calcium.

For detection of calcium mobilization using Fluo-3, cells were cultured on cover slips as described above, and cell-permeant Fluo-3 AM in DMSO was added to 20 µL of medium in the well. The cells were maintained at 22°C for 15 minutes in the Fluo-3 solution. Then, the Fluo-3-containing medium was removed and 20 µL of fresh medium was placed in the well. Optical sections were collected at 30-second intervals for ~5 minutes prior to the treatment, confirming that fluorescence intensity was stable, after which imaging was done for 60 minutes.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Although millions of cells per gram of tumor may enter the bloodstream daily, vanishingly small percentages successfully colonize secondary tissues (7–9). Every step of the metastatic cascade is rate-limiting in that any tumor cell incapable of completing any step is effectively nonmetastatic. Recently, several laboratories have shown that a relatively high proportion of cells entering the vasculature are able to arrive at secondary sites (7). Yet, relatively few respond to the local microenvironment by proliferating. Controlling growth at secondary sites represents a potentially targetable step in the metastatic cascade for therapeutic intervention (10). However, developing agents that block growth at the secondary site requires better biochemical and molecular definition of tumor cell growth (11).

Metastasis suppressors, by definition, block the growth of tumor cells at secondary sites, although still allowing proliferation at the site of tumor development. By inference, many metastasis suppressors are hypothesized to control cellular responses to exogenous signals. This study was undertaken to assess whether signaling pathways implicated in calcium mobilization might be involved in cancer metastasis, specifically cellular suppression of metastasis due to the re-expression of BRMS1. Several laboratories, including ours, previously showed that perturbation of intracellular calcium levels could modify tumor cell invasion and/or metastasis (4, 12, 13). Because phosphoinositides have been implicated in numerous cellular processes involved in tumorigenesis and metastasis, and are key regulators of intracellular calcium (14), we examined phosphoinositide levels in metastatic MDA-MB-435 cells compared with metastasis-suppressed 435/BRMS1 cells.

Phosphoinositide levels were determined by metabolically labeling cells with [3H]myo-inositol, extracting lipids, deacylating the phosphoinositides, and analyzing the resulting glycerophosphoinositols by anion exchange HPLC. Glycerophosphoinositiols from MDA-MB-435 cells were detected in decreasing order of abundance: gPI(4)P > gPI(4,5)P2 > gPI(3)P > gPI(3,5)P2 (Fig. 1C; Table 1). gPI(5)P, gPI(3,4)P2, and gPI(3,4,5)P3 were not detected. Although most of the glycerophosphoinositol levels were similar, a striking difference of gPI(4,5)P2 levels was observed. Figure 1 shows qualitative visual representation comparing MDA-MB-435 and 435/BRMS1 cells. Radioactive counts (cpm) from each gPI(4,5)P2 peak were also measured (Table 1). gPI(4,5)P2 levels in 435/BRMS1 cells averaged between 5% and 10% of those measured for parental MDA-MB-435. Data from Table 1 and Fig. 1 are from the same experiment; however, the data are representative of at least six independent assays.



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  		Figure 1. BRMS1 expression alters phosphoinositide and inositol phosphate levels in MDA-MB-435 human breast carcinoma cells. Cells (435 and 435/BRMS1) were radiolabeled to steady state with [3H]myo-inositol. Radiolabeled phospholipids were extracted, deacylated, and glycerophosphoinositols separated by HPLC. Fractions were collected every 20 seconds and counted in a scintillation counter and cpm plotted. Representative plots(s) for the separation of a broad range (A) or specific (B-D) glycerophosphoinositols is shown for 435 (gray line) and 435/BRMS1 (black line) cells. PtdIns(3,4,5)P3 was not detected in unstimulated 435 and 435/BRMS1 cells. Ins(1,4,5)P3 (E) from 435 (gray line) and 435/BRMS1 (black line) cells was extracted and separated using HPLC and cpm in fractions plotted.

 

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  		Table 1. BRMS1 expression alters PtdIns(4,5)P2 levels in 435/BRMS1 cells

 
The dramatic difference in gPI(4,5)P2 concentrations between MDA-MB-435 and 435/BRMS1 cells could represent PtdIns(4,5)P2 changes at the whole cell level or changes in specific cell structures like the plasma membrane or organelle concentrations. To examine distribution of PtdIns(4,5)P2 in cells, immunofluorescence confocal microscopy with an anti-PtdIns(4,5)P2 monoclonal antibody was done (Fig. 2). Immunofluorescence indicated that PtdIns(4,5)P2 was clearly localized in the plasma membrane, Golgi-like perinuclear structures, and filopodia-like cellular extensions in MDA-MB-435 cells. Nuclear fluorescence was detectable but significantly lower than in other regions of the cells. Although gross morphologies of MDA-MB-435 and 435/BRMS1 are similar, the intensity of anti-PtdIns(4,5)P2 staining was much lower in 435/BRMS1 cells. PtdIns(4,5)P2 was detected only in the perinuclear regions of the 435/BRMS1 cells. No PtdIns(4,5)P2 was observed in cellular extensions, but this may be a consequence of overall reduced staining. Although an extensive panel of assays have not yet been completed for MDA-MB-231 and 231/BRMS1 human breast carcinoma cells, the trends for levels of PtdIns(4,5)P2 and other phosphoinositides are similar (data not shown).



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  		Figure 2. PtdIns(4,5)P2 localization and filamentous actin are altered in 435 cells expressing BRMS1. Immunocolocalization of PtdIns(4,5)P2 in 435 (A) and 435/BRMS1 (B) cells was accomplished using an anti-PtdIns(4,5)P2 monoclonal antibody as described in Materials and Methods. Imaging was done with a laser scanning confocal microscope (excitation, 568 nm; emission, 598 ± 20 nm). Filamentous actin in 435 (C) and 435/BRMS1 (D) cells was stained using phalloidin-Texas red and imaged using confocal microscopy (excitation, 488 nm; emission, 522 ± 16 nm).

 
To begin elucidating the mechanisms by which modulation of PtdIns(4,5)P2 levels might explain BRMS1 suppression of metastasis, the roles of phosphoinositides were considered. The concentrations of Ins(1,4,5)P3 were measured because Ins(1,4,5)P3 is released from PtdIns(4,5)P2 as a result of phospholipase C activity. Metastatic MDA-MB-435 cells contained ~2-fold higher concentrations of Ins(1,4,5)P3 than metastasis-suppressed 435/BRMS1 cells (Fig. 1). The difference, although consistent and biologically significant in many cellular contexts, does not entirely reflect the differences in precursor levels.

Phosphoinositide signaling is considered especially critical in the regulation of intracellular calcium following growth factor stimulation. In response to exogenous signals (e.g., PDGF and insulin), calcium is released from cellular organelles. The calcium acts as a second messenger, mediating a variety of signals. Because we previously found that manipulation of intracellular calcium by chelation with BAPTA-AM (15) could significantly suppress C8161 melanoma cell invasion, motility and metastasis following i.v. inoculation directly into the lateral tail vein of mice,5 intracellular calcium signaling was measured using ratiometric fluorescence with the calcium indicator dye, FURA-2. Monitoring of individual cells revealed generally higher basal activities in MDA-MB-435 cells compared with 435/BRMS1 (Fig. 3); however, the differences were variable and not statistically significant. The differences were pronounced in MDA-MB-435 cells treated with exogenous PDGF (50 ng/mL), a known activator of phosphoinositide signaling; whereas, 435/BRMS1 cell response to PDGF was muted. Control measurements using the calcium ionophore, A23187/ionomycin, showed that intracellular calcium could still be mobilized in 435/BRMS1 cells. It is not known whether PDGF is a key signal for metastatic cells in the lungs in vivo, but our observations highlight the principle that BRMS1 may be mediating one or more signaling pathways from the extracellular milieu to the nucleus. More extensive analyses of suspected growth factors and pathways will be required to ascertain what would be the most relevant to the process of metastatic colonization.



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  		Figure 3. BRMS1 expression reduces the ability of MDA-MB-435 to respond to PDGF. Ratiometric calcium imaging was done on 435 and 435/BRMS1 cells loaded with Fura-2. Cells grown on cover slips were serum-starved for 4 hours, and imaged for 10 minutes prior to stimulation with 50 ng/mL PDGF (dotted vertical line). The Fura-2 excitation ratio of calcium-bound (340 nm) to calcium-free (380 nm) was obtained. *, intracellular calcium stores were released from cells following calcium ionophore (A23187) stimulation in order to confirm cell responsiveness. Tracings are the average of data from 12 separate cells in a representative experiment.

 
PtdIns(4,5)P2 can directly serve as a regulator of vesicle-mediated protein trafficking (16) and as a modulator of cytoskeletal architecture in mammalian cells (5) by binding to effector proteins. Despite the similarity of gross morphology between them, we questioned whether BRMS1-transfected cells might exhibit changes in cytoskeletal architecture. Using fluorophore-tagged phalloidin, consistent changes in filamentous actin levels and organization were observed. But the differences were relatively minor and nondescript.

PtdIns(4,5)P2 plays a pivotal role as a precursor for signaling phosphoinositides. Both precursors of PtdIns(4,5)P2 [PtdIns(4)P, and PtdIns(5)P] and a downstream phosphoinositide [PtdIns(3,4,5)P3] were measured by HPLC. Similar levels were observed in both MDA-MB-435 and 435/BRMS1. Although interexperimental variation was observed, there were no consistent differences noted in detectable phosphoinositides. Because metabolism of the phosphoinositides involves tight regulation of kinase and phosphatase activities, levels of the other molecules may reflect compensatory actions of the enzymes. Direct testing of this hypothesis is not yet possible because the identities of all of the enzymes have not yet been discovered in mammalian cells.

Previous reports have implicated phosphoinositide 3-kinase in metastasis based primarily on experiments using phosphoinositide 3-kinase inhibitors like wortmannin and LY294002, which inhibited invasion in vitro (17). Likewise, loss of the PTEN phosphatase or PTEN function have been correlated with acquisition of metastatic potential (18). Phosphoinositide 3-kinase phosphorylates PtdIns(4,5)P2 to make PtdIns(3,4,5)P3 which, in turn, feeds into the Akt/PKB pathways regulating, among other important phenotypes, apoptosis. Conversely, PTEN acts by removing the 3-phosphate group from PtdIns(3,4,5)P3 to make PtdIns(4,5)P2. Taken together, these observations suggest that BRMS1 might alter metastasis by manipulating PtdIns(3,4,5)P3 via modulation of PtdIns(4,5)P2. Despite numerous attempts to directly test this possibility, PtdIns(3,4,5)P3 could not be routinely detected in either MDA-MB-435 or 435/BRMS1, which is typical of low levels found in cells not stimulated with exogenously added growth factors. Additional experiments are underway to elucidate PtdIns(3,4,5)P3 induction in BRMS1-transfected cells treated with growth factors; however, the data to date do not definitively support or refute a role for PtdIns(3,4,5)P3 in BRMS1-mediated suppression of breast cancer metastasis.6

Despite significant improvements in the understanding of cellular growth control and the perturbations involved in developing tumors, the mechanisms that regulate tumor cells' abilities to grow in tissues other than those from which they originated (i.e., metastasis) are still largely unknown. The discovery of metastasis suppressors, the demonstration that many metastasis suppressors seem to block metastasis by interfering with sustained cell proliferation at the secondary site, and the availability of matched metastatic and nonmetastatic cell lines now enable researchers to design experiments that will aid in the understanding of the important biological phenomenon of metastatic colonization. The dissemination of cancer cells from the primary tumor is apparently not infrequent. However, the colonization of tissues by disseminated cells fortunately is rare. If the mechanisms of action for metastasis suppressors, like BRMS1, can be understood at a molecular level, then it may be possible to intervene and reduce morbidity and mortality due to cancer metastasis. The findings of this report suggest that BRMS1 suppresses metastasis by modulation of phosphoinositide signaling, specifically by controlling the levels of PtdIns(4,5)P2.


   Acknowledgments
 
Grant support: Supported primarily by a grant from the National Foundation for Cancer Research, with additional support from USPHS grants RO1-CA87728, P50-CA89019, and RO1-NS029632.

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 Dr. Glenn Prestwich (University of Utah) for thoughtful comments and suggestions. We are also appreciative to members of both the DeWald and Welch laboratories for their advice and comments.


   Footnotes
 
Note: Current affiliation, Metastasis and Tumor Biology Research Center, University of South Alabama, Mobile, AL.

5 D.R. Welch, unpublished observations. Back

6 D.B. DeWald and D.R. Welch, unpublished observations. Back

Received 8/31/04. Revised 10/21/04. Accepted 12/ 1/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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  2. Meehan WJ, Welch DR. Breast cancer metastasis suppressor 1: update. Clin Exp Metastasis 2003;20:45–50.[CrossRef][Medline]
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D. G. Hicks, B. J. Yoder, S. Short, S. Tarr, N. Prescott, J. P. Crowe, A. E. Dawson, G. T. Budd, S. Sizemore, M. Cicek, et al.
Loss of Breast Cancer Metastasis Suppressor 1 Protein Expression Predicts Reduced Disease-Free Survival in Subsets of Breast Cancer Patients.
Clin. Cancer Res., November 15, 2006; 12(22): 6702 - 6708.
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Z. Zhang, H. Yamashita, T. Toyama, Y. Yamamoto, T. Kawasoe, and H. Iwase
Reduced expression of the breast cancer metastasis suppressor 1 mRNA is correlated with poor progress in breast cancer.
Clin. Cancer Res., November 1, 2006; 12(21): 6410 - 6414.
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D. Datta, J. A. Flaxenburg, S. Laxmanan, C. Geehan, M. Grimm, A. M. Waaga-Gasser, D. M. Briscoe, and S. Pal
Ras-induced Modulation of CXCL10 and Its Receptor Splice Variant CXCR3-B in MDA-MB-435 and MCF-7 Cells: Relevance for the Development of Human Breast Cancer
Cancer Res., October 1, 2006; 66(19): 9509 - 9518.
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