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The Breast Cancer Metastasis Suppressor 1 (BRMS1) belongs to an expanding category of proteins called metastasis suppressors that demonstrate in vivo metastasis suppression while still allowing growth of the orthotopic tumor. Since BRMS1 decreases either the expression or function of multiple mediators implicated in resistance to chemotherapy (NF-κB, AKT, EGFR), we asked whether breast carcinoma cells expressing BRMS1 could be sensitized upon exposure to commonly used therapeutic agents that inhibit some of these same cellular mediators as BRMS1. In this report, we demonstrate that chemosensitivity of breast cancer cells is preserved in the presence of BRMS1. Further, BRMS1 does not change expression of AKT isoforms or PTEN, implicated in chemoresistance to common drug agents. Overall, our data with two different metastatic breast cancer cell lines indicates that BRMS1 expression status may not interfere with the response to commonly used chemotherapeutic agents in the management of solid tumors such as breast cancer. Since tumor protein expression analysis increasingly guides therapy decisions, our data may be of clinical benefit in disease management including profiling for BRMS1 expression before start of therapy.
The Breast Cancer Metastasis Suppressor 1 (BRMS1) belongs to an expanding class of proteins called metastasis suppressors that demonstrate in vivo metastasis suppression while allowing growth of the orthotopic tumor [1–3]. BRMS1 functions as a metastasis suppressor in animal models of breast , melanoma , ovarian carcinomas . Recent studies with clinical samples have indicated a correlation between loss of BRMS1 expression and poor prognosis in a subset of patients [7–9]. Experimentally, loss of metastasis suppressors, including BRMS1 may be reversed using therapeutic agents [10, 11] suggesting use of BRMS1 and other metastasis suppressors as markers and a potential adjuvant role of such “re-expression therapy” in the management of metastasis. Experimentally, BRMS1 expression increases susceptibility to anoikis which is proposed to contribute, in part, to metastasis suppression [12, 13]. BRMS1 is part of the Sin3-HDAC chromatin remodeling complexes [14, 15] that regulate gene expression and which could potentially alter chemotherapeutic responses . Consequently, BRMS1 regulates expression of several signaling intermediates including epidermal growth factor receptor , osteopontin [18, 19], phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) , urokinase plasminogen activator , fascin , and connexins . Further, BRMS1 regulates nuclear factor-kappa B (NF-κB) activity  and AKT phosphorylation  in response to exogenous stimuli implicated in chemoresistance in a number of cancer models [23–25]. Recently, Rivera and colleagues suggested that BRMS1 expression may increase chemosensitivity as a consequence of downregulation of 14-3-3-γ, sorcin, and Hsp27 .
Taken together, since BRMS1 decreases either the expression or activity of multiple mediators implicated in resistance to chemotherapy (e.g. NF-κB, AKT, EGFR) and increases susceptibility to anoikis, we asked whether breast carcinoma cells expressing BRMS1 could respond differently upon exposure to commonly used therapeutic agents in the treatment of breast cancer. In this report, using multiple approaches we evaluated that chemosensitivity of breast cancer cells is preserved in the presence of BRMS1. Further, BRMS1 does not change expression of AKT isoforms or PTEN, implicated in chemoresistance to common drug agents. Information from these studies may be potentially used in the clinic in stratifying patients and designing treatment courses in the management of metastatic disease.
MDA-MB-231 and MDA-MB-435 breast adenocarcinoma cells  were transfected with a lentiviral vector construct expressing BRMS1 under the control of a cytomegalovirus promoter . MDA-MB-231/435 vector transfectants (231/435), and 231BRMS1/435BRMS1 were cultured in a 1:1 mixture of Dulbecco’s-modified essential medium (DMEM) and Ham’s F-12 medium supplemented with 1% non-essential amino acids, and L-glutamine (Invitrogen, Carlsbad, CA) and containing 5% fetal bovine serum (cDMEM-F12). 231 and 231BRMS1 cells were passaged using 0.125% trypsin and 2 mM EDTA solution (Invitrogen, Carlsbad, CA) and 435 and 435BRMS1 cells were passaged using 2 mM EDTA in Ca2+/Mg2+- free PBS. Cell lines were confirmed to be free of Mycoplasma contamination using PCR (TaKaRa, Japan). No antibiotics or antimycotics were used.
Doxorubicin, vincristine were dissolved in water and 5-fluorouracil (5-FU), paclitaxel were dissolved in dimethyl sulfoxide. Stock solutions of doxorubicin (10 mM), vincristine (1 mM) were stored at 4 C and 5-FU (500 mM), paclitaxel (1 mM) were stored at −20°C according to manufacturer’s instructions. For final drug concentrations, solutions were serially diluted in media and added to wells. The highest doses of doxorubicin, vincristine, 5-FU, and paclitaxel used for assays were 20 μM, 1 μM, 2000 μM and 1 μM respectively. All drugs were purchased from Sigma-Aldrich, St. Louis, MO and were used within one week of preparation.
Cells (231/231BRMS1 and 435/435BRMS1) were passaged and allowed to proliferate to 70% confluence in 10 cm dishes for at least 2 passages to ensure log growth before harvesting for seeding. Cells were seeded in triplicate at a density of 1000 cells/well onto 6-well plates (Corning) in a final volume of 2 ml media and allowed to attach overnight. The following day, drugs were added at the indicated final concentrations in a volume of 2 ml media and incubated with cells for 4 h. Drug containing media was aspirated gently after 4 h and replaced with 5 ml fresh pre-warmed medium in each well . Cells were left undisturbed in a humidified incubator at 37°C for 7 days and colonies formed were counted by staining with a 1:3 solution of acetic acid:methanol containing 0.01% crystal violet (Carnoy’s fixative). Experiments were performed in triplicate and repeated at least twice independently.
Exponentially growing 231/231BRMS1 and 435/435BRMS1 cells were plated at a density of 2500 cells/well in 24-well plates in quadruplicate and allowed to attach overnight. The next day, drugs were added at the indicated concentrations in a final volume of 500 μl and cells were exposed to drugs for 4 h. After 4 h, drug containing media was gently aspirated and replaced with 1 ml of fresh medium and cells were incubated undisturbed in a humidified incubator until the time of assay. Every second day beginning the day of drug addition, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO) was added to the media in each well at a final concentration of 0.5 mg/ml and incubated with cells for 3 h. Following incubation with MTT, medium was aspirated gently and 500 μl of DMSO was added to each well and plates were shaken on a horizontal shaker for 30 minutes to dissolve the formazan crystals. Absorbance was read in a plate reader at 490 nm and experiments were repeated independently at least twice.
Exponentially growing 231/231BRMS1 and 435/435BRMS1 cells were plated at a density of 5000 cells/well on 8-well chamber slides (Nunc, Nalgene). Before plating cells, each well was coated with a Matrigel cushion (40 μl). The final concentration of Matrigel above the cushion layer was adjusted to 10%. Media containing cells (200 μl) was mixed with a 20% Matrigel suspension in cold media containing drug at 2X of the indicated concentrations (200 μl). The two suspensions were mixed together gently to ensure equal distribution of cells and drugs and incubated in a humidified incubator at 37°C. Cells were incubated for nine days and colonies formed were counted. Experiments were repeated independently at least twice.
Cells grown to approximately 80% confluence on 100 mm dishes were rinsed 2X with ice-cold PBS cells 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 cocktail containing aprotinin, leupeptin, and phenylmethylsulfonyl fluoride (Roche, Indianapolis, IN). Protein concentration was determined using a Bradford colorimetric assay (Pierce, Rockford, IL). Protein was denatured with Laemmli’s buffer at 95°C for 5 min and lysate equal to 50 μg total protein was loaded onto each well. Proteins were separated using either 8% or 12% SDS-PAGE gels and resolved proteins were transferred to PVDF 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 AKT1, AKT2, AKT3, PTEN (Cell Signaling, Danvers, MA) overnight at 4°C and subsequently with HRP-conjugated secondary antibody at room temperature for 2 h. Gels were either run separately for each protein or membranes were stripped with stripping buffer (Pierce, Rockford, IL) and re-probed. Signals were visualized using ECL (Amersham Biosciences, Piscataway, NJ) following manufacturer’s instructions. Blots were probed with an anti-β-actin antibody (Sigma, St. Louis, MO) to confirm equal protein loading.
Clonogenic assays were used to determine dose-responses over a minimum of four log concentration range for each drug with 231/231BRMS1 and 435/435BRMS1 cells. IC50 values and corresponding confidence intervals were determined from the dose-response curves generated using Prism software (GraphPad Software, La Jolla, CA) (Fig. 1, Table 1). A trend towards BRMS1-expressing cells forming fewer colonies was observed with most drugs in 231 as well as 435 cells, although statistical significance was not reached. IC50 values were similar in BRMS1-expressing vs. vector transfected cells (Fig. 1, Table 1).
Consistent with previous results, expression of BRMS1 did not affect proliferation of either 231 or 435 cells [4, 13]. Addition of chemotherapeutic agents inhibited the proliferation of vector transfected cell lines in a dose-dependent manner as measured by MTT assays. Following exposure to chemotherapeutic agents, BRMS1 expressing cells showed a dose-dependent decrease in proliferation that paralleled decreases observed with the vector transfectants (Fig. 2).
Cells grown in 3D-culture exhibit distinct structural, behavioral phenotypes and drug responses compared to 2D-culture [29–31]. Further, responses to chemotherapy appear to be distinct in 2D- vs. 3D-culture . Therefore, we tested whether the response of BRMS1- expressing cells to chemotherapeutic agents was modified under 3D-culture conditions. Consistent with observations that cells in 3D-culture display differential drug responses [29–31], IC50 values obtained were different from those obtained in clonogenic assays (Fig. 3A, Table 1, and Table 2). A dose-dependent decrease in colony formation was observed in 435 and 435BRMS1 cells. BRMS1-expressing cells tended to form fewer colonies in 3D-culture compared to 435 cells, however, the results were variable and IC50 differences did not reach statistical significance (Fig. 3A, Table 2). Similar results were obtained with 231 and 231BRMS1 expressing cells (Table 2). Although not quantified, colony sizes between vector transfectants and BRMS1-expressing cells with or without addition of chemotherapeutic agents were also not appreciably different, although colony size decreased with increasing drug concentrations.
Since chemoresistance has been correlated with increased expression of AKT, expression of AKT isoforms in vector-transfected vs. BRMS1-expressing cells was tested by immunoblotting. Basal protein levels of AKT1 and AKT2 did not change significantly in BRMS1 expressing cells or changes were not consistent between cell lines (Fig. 3B). AKT3 was undetectable in either cell line or BRMS1-expressing cells. Since BRMS1-expressing cells have >95% lower levels of PtdIns(4,5)P2 , we also examined whether PTEN (catalyzes conversion of PtdIns(3,4,5)P2 to PtdIns(4,5)P2) expression was altered by BRMS1 expression. PTEN protein expression was unchanged in BRMS1 expressing cells.
Innate or acquired chemoresistance to therapeutic agents remains a challenge in the management of cancer and especially metastasis. Multi-drug therapy is often targeted at the heterogeneous residual cancer cells, usually following surgical resection, with the hope of preventing recurrence. In addition to standard-of-care drugs, the choice of particular chemotherapeutics is often guided by histo- and/or expression analysis of various markers -dependent on cancer type . Expression profiling of tumors before initial chemotherapy is becoming increasingly common and has aided in the development of targeted therapy including Herceptin® and Avastin®.
Histochemical analysis reveals that clinically, loss of BRMS1 is correlated with poor prognosis in a subset of breast carcinomas [7–9]. Further, BRMS1 expression is lost in higher grade ovarian  and supraglottic laryngeal carcinomas . At the molecular level, increased AKT and NF-κB activity have been demonstrated to contribute to chemoresistance [23–25]. BRMS1 selectively decreases AKT phosphorylation in response to growth factors  and downregulates NF-κB activity . Although we show here that BRMS1 does not modulate PTEN expression, PtdIns(4,5)P2 is decreased by >95% in BRMS1-expressing cells . Since BRMS1 downregulates a number of mediators of chemoresistance, and since BRMS1 expression appears to be important in determining metastatic fate, these data provided a compelling rationale to test the hypothesis that BRMS1 would increase chemosensitivity of breast cancer cells. To our surprise, BRMS1 expression did not alter chemosensitivity of breast cancer cells in multiple in vitro assays.
Chemotherapeutic agents in this study were selected based on their known mechanisms of action, and their ability to antagonize or act through some of the same pathways mediated by BRMS1. For example, doxorubicin has been demonstrated to decrease levels of PtdIns(4,5)P2 while vincristine mediates cell death, in part, by activation of pro-apoptotic genes through NF-κB [36, 37]. Previous data indicates that BRMS1 expression did not alter in vitro proliferation or rate of in vivo tumor growth [4, 13, 38, 39]. Halogenated pyrimidines including 5-FU are metabolized to derivatives that replace thymidylate in actively dividing cells . The anthracycline antibiotic doxorubicin (adriamycin) forms a trimer-complex with topoisomerase-II and DNA and also intercalates with DNA in dividing cells preventing DNA replication. The relative lack of effect of 5-FU and doxorubicin on both colony formation and proliferation of BRMS1-expressing cells may therefore be explained by the lack of change in proliferation rate by BRMS1 in vitro, although a comparable dose-dependent decrease in assay end-points is observed. Doxorubicin is also reported to decrease PtdIns(4,5)P2 levels  (as BRMS1 does), however, no potentiation of growth inhibition was observed when BRMS1-expressing cells were exposed to doxorubicin. The vinca alkaloid vincristine and the diterpenoid paclitaxel both bind β-tubulin and disrupt microtubule polymerization and disassembly respectively. BRMS1 has recently been shown to downregulate β-tubulin 6 (TUBB6) expression by microarray analysis . In the presence of downregulation of TUBB6, BRMS1-expressing cells show no apparent disruption in cell-cycle progression (data not shown). Inhibition of NF-κB is known to sensitize tumor cells to microtubule disrupting agents such as paclitaxel and vincristine . Reports also indicate that activation of NF-κB following vincristine exposure is perhaps required for inducing pro-apoptotic genes [36, 37]. The apparent lack of change in survival of BRMS1-expressing cells may reflect antagonistic signaling through NF-κB. Expression analysis of common mediators of chemoresistance including AKT isoforms and PTEN also did not show consistent changes in BRMS1-expressing cells suggesting that the lack of increased sensitivity to drugs tested may not involve these mediators.
Overall, our data with two different breast cancer cell lines indicates that BRMS1 expression status does not modify the response to commonly used chemotherapeutic agents although the mechanisms of actions of these drugs and BRMS1 intersect at various levels of signaling. Since tumor protein expression analysis increasingly guides therapy decisions, and since loss of BRMS1 is predictive of metastatic outcome , our data may be of translational benefit in directing the course of disease management including profiling for BRMS1 expression before start of therapy.
This work was supported by US Public Health Service Grant (R01-CA87728) with additional support from the National Foundation for Cancer Research - Center for Metastasis Research (DRW). KSV was supported by a postdoctoral fellowship (PDF1122006) from Susan G Komen For the Cure. JSS was supported by the Program for Research Experience in Pathology (P.R.E.P.) at the University of Alabama at Birmingham. We thank members of the Welch laboratory for their helpful suggestions and discussion and Dr. Monica M. Richert for technical assistance with the 3D-culture system.
Conflict of Interest statement
The authors declare no conflicts of interest.
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