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Expression of SCC-S2, an antiapoptotic molecule, correlates with enhanced proliferation and tumorigenicity of MDA-MB 435 cells

Abstract

SCC-S2/GG2-1/NDED is a recently discovered antiapoptotic molecule induced by the activation of the transcription factor NF-κB. Here we have examined a role of SCC-S2 in cell growth regulation in vitro and in vivo. Western blotting using an antipeptide antibody revealed endogenous SCC-S2 as a 21 kDa cytosolic protein in human breast cancer cells (MDA-MB 231) and renal carcinoma cells (RCC-RS). The immunofluorescence detection method showed the cytosolic localization of FLAG-tagged human SCC-S2 in COS-1 transfectants. MDA-MB 435 human cancer cells stably transfected with the FLAG-tagged SCC-S2 cDNA exhibited increased growth rate as compared to control vector transfectants, as measured by the cell viability (>twofold; n=3; P<0.005) and thymidine-labeling procedures (sixfold; n=3; P<0.0001). SCC-S2 transfectants also displayed an increase in cell migration in collagen I as compared to control transfectants (twofold; n=3; P<0.005). In athymic mice, SCC-S2 transfectants showed significantly enhanced tumor growth as compared to control transfectants (mean tumor volumes, day 16: control, 56.86±19.82 mm3; SCC-S2, 127.54±18.78 mm3; n=5; P<0.03). The examination of a limited number of clinical specimens revealed higher expression levels of SCC-S2 protein in certain human tumor tissues as compared to the matched normal adjacent tissues. Taken together, the present studies demonstrate SCC-S2 as a novel oncogenic factor in cancer cells.

Main

Previously, we have reported the identification and characterization of SCC-S2, a novel 21 kDa antiapoptotic protein (Patel et al., 1997; Kumar et al., 2000). SCC-S2/GG2-1/NDED contains one death effector domain (DED)-like domain homologous to DED II of Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE/caspase 8)-inhibitory proteins (FLIPs) (Horrevoets et al., 1999; Kumar et al., 2000; You et al., 2001). The functional significance of a DED-like domain in SCC-S2 is not known. c-FLIPL contains two DEDs (I and II) and an inactive caspase domain (Irmler et al., 1997). Death domain (DD)-containing receptor-mediated apoptosis is blocked by FLIP, which can interact via its DEDs with the adapter molecule Fas-associated death domain (FADD) and caspase 8, preventing the activation of caspase 8 (Eberstadt et al., 1998; Wang et al., 2000; Yeh et al., 2000; Thome and Tschopp, 2001; Kaufmann et al., 2002; Xiao et al., 2003). Additional studies suggest a link between the DED-containing proteins and mitochondrial pathways of cell death or cell survival (Kuwana et al., 1998; Hackam et al., 2000; Zhang et al., 2000). We and others have shown that SCC-S2/NDED mRNA expression is induced by TNF-α and by activation of the transcription factor NF-κB (Kumar et al., 2000; You et al., 2001). In addition, overexpression of SCC-S2/NDED is associated with enhanced survival and inhibition of activities of the apoptotic enzymes caspase 8 and caspase 3 (Kumar et al., 2000; You et al., 2001). Cell survival and malignant growth-related signaling pathways are intricately linked (Owen-Schaub et al., 1998; Wajant, 2002; Tibbetts et al., 2003). Accordingly, we reasoned that SCC-S2 may play a role in the regulation of cell growth and proliferation. The main purpose of this study was to determine the subcellular localization of SCC-S2, and to examine a possible function of SCC-S2 in tumor cell growth in vitro and in vivo.

Polyclonal antibodies were generated against three peptides (P1, P2, and P3) derived from the amino-acid sequence of SCC-S2 (Figure 1a). The anti-SCC-S2 antibody recognized a single band at approximately 21 kDa in human breast cancer cells (MDA-MB 231 and MDA-MB 435) and renal carcinoma cells (ACHN and RCC-RS) (Figure 1b, left panel; data not shown). The corresponding preimmune serum (PI) was used as a negative control. Subcellular fractionation and Western blotting procedures indicated that endogenous SCC-S2 is a cytosolic protein in human breast (MDA-MB-231) and renal carcinoma cells (RCC-RS) (Figure 1b, right panel). In transient transfection studies, HeLA cells or COS-1 cells transfected with an expression vector containing FLAG epitope-tagged human SCC-S2 cDNA revealed a 21 kDa band (Figure 1c). A diffused cytosolic/perinuclear distribution of the FLAG-tagged SCC-S2 protein in COS-1 transfectants was observed by the indirect immunofluorescence assay (Figure 1d).

Figure 1
figure1

Intracellular localization of SCC-S2. (a, b) SCC-S2 is a 21 kDa cytosolic protein. The amino-acid sequence of SCC-S2 has been reported earlier (Kumar et al., 2000). Rabbit polyclonal antibodies, anti-P1, anti-P2, and anti-P3 were generated against peptides P1, P2, and P3, respectively (panel a). Peptides were synthesized and custom antibodies were developed at the commercial facilities (P1, Zymed Laboratories Inc.; P2 and P3, Covance Research Products Inc.). MDA-MB 231 human breast cancer cells or RCC-RS human renal carcinoma cells (Leung et al., 1993) were cultured in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 25 μg/ml gentamicin in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Whole-cell lysates (panel b, right), and cytosol- and nuclear-enriched fractions (panel b, left) were prepared as described before (Kumar et al., 2000; Isaacs et al., 2001), followed by electrophoresis and immunoblotting with the indicated polyclonal anti-SCC-S2 antiserum. The blots were reprobed with antiactin antibody (SIGMA), or antitubulin antibody (Santa Cruz). PI, the corresponding preimmune serum; IB, immunoblot; C, cytosolic fraction; N, nuclear-enriched fraction. (c) Transient transfection and expression of FLAG-tagged SCC-S2 cDNA. HeLa or COS-1 cells were transiently transfected with pCR3.1FLAG-tagged SCC-S2 cDNA or vector pCR3.1 using LipofectAMINE 2000 (Invitrogen), as described before (Kumar et al., 2000). SCC-S2 protein expression was detected in HeLa transfectants by immunoblotting with anti-SCC-S2 antiserum (anti-P3), followed by reprobing with mouse monoclonal anti-FLAG M2 antibody (SIGMA). SCC-S2 protein expression was detected in COS-1 transfectants by immunoblotting with anti-FLAG antibody. The blots were reprobed with antiactin antibody or anti-GAPDH antibody. (d) Detection of SCC-S2 expression by immunofluorescent staining. COS-1 cells were grown on coverslips and transiently transfected with FLAG-tagged SCC-S2 cDNA (SCC-S2) or vector pCR3.1 (PCR3.1) using LipofectAMINE 2000. A modification of the indirect immunofluorescence procedure (Mueller et al., 1992) was used. SCC-S2 protein was detected using mouse monoclonal anti-FLAG M2 antibody (SIGMA), followed by Alexa Fluor 488 donkey anti-mouse secondary antibody (Molecular Probes). The nuclei were counterstained with 4′,6-diamidino-2-phenyl-indole dihydrochloride (DAPI). The coverslips were mounted onto glass slides using Fluoromount-G (Southern Biotechnology Associates, Inc.), and cells were imaged using the Nikon E600 Fluorescence Microscope System. Top right, anti-FLAG; top left, DAPI; bottom right, merge; and bottom left, anti-FLAG

To determine the effect of the expression of SCC-S2 on cell growth and proliferation, MDA-MB 435 cells were stably transfected with FLAG-tagged SCC-S2 cDNA containing the coding region of SCC-S2 or pCR3.1 expression vector DNA. Northern blot analysis revealed a 650 bp band corresponding to the exogenous transcript in SCC-S2 transfectants. In addition, an 1.9 kb endogenous transcript was seen in SCC-S2 and vector transfectants (Figure 2a). Expression of SCC-S2 protein was confirmed by immunoblotting with anti-FLAG antibody (Figure 2b). SCC-S2 transfectants exhibited profound changes in morphology and growth rate as compared to vector control transfectants. Overexpression of SCC-S2 correlated with distinct spindle-shaped morphology and increase in anchorage-independent growth revealed by increased foci formation in culture (Figure 2c). At 96 h after seeding, the number of SCC-S2 transfectants was 2–3-fold higher than the vector population (P<0.005, n=3, Figure 2d). Consistent with these observations, SCC-S2 transfectants showed a 5–7-fold increase in DNA synthesis as compared to vector transfectants (P<0.0001, n=3) (Figure 2e).

Figure 2
figure2

Expression of SCC-S2 correlates with enhanced proliferation and motility of MDA-MB 435 human breast cancer cells. (a, b) Stable transfection and expression of SCC-S2 cDNA. MDA-MB 435 cells were grown in 75-cm2 tissue culture flasks in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 25 μg/ml of gentamicin (all from Invitrogen Life Technologies, Inc.). For stable transfections, 10 μg of plasmid DNA (pCR3.1SCC-S2-FLAG or pCR3.1 vector) was resuspended in 500 μl of Optimem medium (Invitrogen Life Technologies, Inc) and mixed with 60 μl of LipofectAMINE in 500 μl of Optimem medium. The mixture was incubated at room temperature for 45 min and added to a 70–80% confluent T-75 flask. After 48 h, the cells were trypsinized, pooled, and cultured in DMEM containing 10% FBS and 800 μg/ml of G418 (Invitrogen Life Technologies). Approximately 100 G418-resistant colonies were pooled and stable transfectants were maintained in DMEM supplemented with 10% FBS, 25 μg/ml gentamicin, and 600 μg/ml of G418. The SCC-S2 mRNA expression was detected by Northern blot analysis using radiolabeled SCC-S2 cDNA as probe, and the blot was reprobed with radiolabeled β-actin cDNA, as described before (Kumar et al., 2000) (panel a). Expression of SCC-S2 protein was detected by Western blotting using M2 mouse anti-FLAG antibody (SIGMA) (panel b). (c) Altered morphology of the MDA-MB 435-SCC-S2 transfectants. The stably transfected cells were plated as monolayers in T-25 flasks and allowed to grow for 6 days. Photographs were taken using Nikon SMZ-1500 EPI-Fluorescence Stereoscope System (top panels, × 20; bottom panels, × 100). (d) MDA-MB 435-SCC-S2 transfectants show enhanced growth rate in vitro. Stable transfectants were seeded in six-well plates (50 000 cells/well, seeded in triplicate per time point, total five time points). The initial cell count was determined at 24 h. The medium was aspirated and cells were trypsinized and counted using the trypan blue dye-exclusion method. Cell numbers were similarly determined at 48, 72, and 96 h, and fold increase in cell number at each time point versus the initial count was plotted. Data obtained from a representative of two independent experiments are shown. (e) SCC-S2 expression correlates with increased DNA synthesis. MDA-MB 435 stable transfectants were seeded in triplicate into 24-well plates (50 000 cells/well). At 24 h, the medium was changed to a fresh medium containing 1 μCi/ml [3H]thymidine (86.9 Ci/mmol) (Perkin-Elmer) for an additional 6 h. The DNA synthesis was measured as trichloroacetic acid-precipitable radioactivity as described (Lim et al., 2002). Data from a representative of three experiments, each performed in triplicate, are shown. (f) MDA-MB 435-SCC-S2 transfectants display enhanced migration on collagen-I. A modification of the Boyden Chamber assay (Adelsman et al., 1999) was used to compare the migration potential of MDA-MB 435-SCC-S2 and pCR3.1 vector stable transfectants. In brief, the underside of the polycarbonate membrane of the upper chamber was coated either with 20 mg/ml of rat tail collagen-I (UBI) or 1.5% BSA (SIGMA) as control for 2 h at 37°C. MDA-MB 435 transfectants were serum-starved overnight, trypsinized, washed twice with migration buffer (Fibroblast basal medium, FBM, Clonetics) supplemented with 1.5% BSA (FBM/BSA) and resuspended in the same buffer at a concentration of 1 × 106 cells/ml. For the migration assay, 150 μl of the cell suspension was added to each Boyden chamber and the bottom chamber was filled with 300–400 μl FBM/BSA. The migration assay was performed at 37°C for 5–6 h. Nonmigratory cells at the upper membrane surface were removed with several cotton swabs, and the migratory cells at the bottom of the membrane were stained with crystal violet stain (0.1% crystal violet in 0.1 M borate buffer, pH 9.2, and 2% ethanol) for 20 min at room temperature. The stain was extracted with 10% acetic acid; an aliquot of the stained solution was used to measure the optical density at 600 nm. The experiments were performed in triplicate and all readings have had their background values subtracted. The data shown are the mean of two representative experiments each performed in duplicate or triplicate

SCC-S2 was first identified based on its relatively higher mRNA expression in a head and neck squamous carcinoma cell line established from a metastatic tumor, as compared to the head and neck squamous carcinoma cell line established from the matched primary tumor (Patel et al., 1997). Motility of tumor cells is an important parameter of their aggressive growth and the malignant progression phenotype (Keleg et al., 2003). We asked if stable overexpression of SCC-S2 causes changes in the cell migration characteristic of the MDA-MB 435 transfectants. As shown in Figure 2f, SCC-S2 transfectants showed a modest increase in cell migration on collagen-I as compared to vector transfectants (1.5–2-fold; P<0.005; n=3). These results suggest that SCC-S2 expression promotes in vitro growth, proliferation, and motility of MDA-MB 435 cells.

We next examined the effect of SCC-S2 expression on MDA-MB 435 tumor growth in athymic mice. The tumors were palpable in the first week after inoculation of cells into the mammary fat pads of female athymic mice. As shown in Figure 3a, SCC-S2 transfectants revealed a remarkably accelerated tumor growth as compared to vector transfectants throughout the observation period. The mean tumor volumes of SCC-S2-transfected cells were significantly greater than control transfectants (day 16: control, 56±19.82 mm3; SCC-S2, 127.54±18.78 mm3; n=5; P<0.03). The animals were killed in accordance with the institutional tumor burden guidelines, and tumors were excised. The expression of exogenous SCC-S2 protein was detected in tumor tissues by immunoprecipitation, followed by immunoblotting using anti-FLAG antibody (Figure 3b). These data demonstrate that overexpression of SCC-S2 causes an increased malignant growth of MDA-MB 435 cells in vivo.

Figure 3
figure3

MDA-MB 435-SCC-S2 transfectants exhibit enhanced tumor growth in athymic mice. (a) Female athymic mice (8-week old; BALB/c nu/nu) were obtained from the National Cancer Institute (Fredrick, MD, USA), and acclimatized for 1 week. Mice were fed a diet of animal chow and water ad libitum. Logarithmically growing MDA-MB 435 transfectants (SCC-S2 or control vector) were injected subcutaneously into the mammary fat pads of mice (0.5 × 106 cells/animal). The viability of the cells prior to inoculation was determined by the trypan blue dye exclusion method. The tumor volumes were measurable by day 6 postinoculation. Tumor volumes were determined from caliper measurements of the three major axes (a,b,c) and calculated using abc/2, an approximation for the volume of an ellipse (πabc/6). Mean tumor volume±standard error (s.e.) was plotted. ,Analysis of variance (one-way ANOVA) was performed to determine the statistical significance of changes in tumor volumes observed on day 16 postinoculation. (b) Expression of FLAG-tagged SCC-S2 in tumor xenografts. The expression of exogenous SCC-S2 in tumor tissues was detected by immunoprecipitation (IP), followed by immunoblotting (IB) using anti-FLAG antibody

As a step towards further validation of a role of SCC-S2 in tumor progression, we compared the levels of SCC-S2 protein expression in a limited number of human normal tissues and matched breast or renal cell carcinoma tissues. SCC-S2 protein expression was found to be higher (approximately 2–31-fold) in several breast cancer samples as compared to normal adjacent tissues examined (6/10; patients nos. 2, 3, 5, 6, 7, and 10) (Figure 4a). In one patient (no. 1), SCC-S2 expression was higher in the normal tissue. In the remaining three patients (nos. 4, 8, and 9), SCC-S2 protein expression levels were comparable in the normal and matched tumor tissues (Figure 4a). SCC-S2 expression was also higher (approximately 2–68-fold) in most of the renal cell carcinoma samples as compared to normal adjacent tissues examined (6/9; patients nos. 2, 4, and 6–9) (Figure 4b). In the tissue lysates, anti-SCC-S2 antibody appeared to crossreact with a protein slightly higher in molecular weight than SCC-S2. This additional band was not seen in the lysates from several tumor cell lines tested (data not shown). The SCC-S2 isoforms have been identified (GenBank accession number AF099935; DK and UK, unpublished data) and further studies are required to determine the nature of this higher molecular weight band detectable in tissue samples. Increased expression of SCC-S2 in 63% of the breast and renal tumor tissues examined (n=19) is consistent with enhanced proliferation of MDA-MB 435 SCC-S2 transfectants in vitro and in vivo, and supports a role of SCC-S2 in carcinogenesis.

Figure 4
figure4

SCC-S2 expression in human tumor and normal adjacent tissues. Histologically confirmed frozen human tissues from breast cancer patients (a) and renal cell carcinoma (RCC) patients (b) were obtained from Co-operative Human Tissue Network (CHTN) resource of the National Cancer Institute (NIH). The tissues were homogenized in RIPA lysis buffer (NaCl, 150 mM, pH 7.5; sodium deoxycholate, 1% (w/v); Triton X-100, 1% v/v; SDS, 0.1% w/v) containing 2 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (20 μg/ml), and leupeptin (20 μg/ml). After incubation on ice for 30 min, the tissue extracts were centrifuged at 12 000 g for 15 min at 4°C, followed by SDS–PAGE and Western blotting using anti-SCC-S2 antibody. The blots were reprobed with antiactin antibody. The blots were scanned and bands were quantified and normalized against the actin signal using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). The histograms show fold changes in SCC-S2 protein expression in tumor tissues (solid bars) relative to normal adjacent tissues examined (empty bars)

In conclusion, the present data demonstrate that SCC-S2 is an oncogenic factor that may play a role in tumor progression. The mechanisms of SCC-S2-mediated cell proliferation and oncogenesis are as yet unknown. Exogenous expression of SCC-S2 enhances cell survival and appears to correlate with the G1-to-S phase transition (Kumar et al., 2000; DK and UK, unpublished data). As mentioned earlier, activation of NF-κB leads to enhanced expression of SCC-S2 (Kumar et al., 2000; You et al., 2001). Constitutive upregulation of NF-κB has been reported in a variety of cancers (Bargou et al., 1997; Nakshatri et al., 1997; Sovak et al., 1997; Mori et al., 1999; Wang et al., 1999b; Darnell, 2002; Orlowski and Baldwin, 2002; Scaife et al., 2002; Nair et al., 2003). In addition, NF-κB-inducible genes, including the antiapoptotic protein cFLIP, urokinase-type plasminogen activator (uPA), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and cyclin D1 have been associated with tumor progression (Irmler et al., 1997; Djerbi et al., 1999; Hinz et al., 1999,2002; Medema et al., 1999; Wang et al., 1999a; Micheau et al., 2001; Ryu et al., 2001). One possibility is that SCC-S2 may represent an effector of the NF-κB signal transduction pathway regulating cell survival and proliferation. Mechanistic and preclinical studies are currently underway in our laboratory in an attempt to ultimately establish SCC-S2 as a potential therapeutic target.

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Acknowledgements

MDA-MB 231 and MDA-MB 435 human cancer cells were obtained from the Tissue Culture Shared Resource facility of the Lombardi Cancer Center, Georgetown University Medical Center. Mice studies were performed at the Research Resource Facility of the Division of Comparative Medicine, Georgetown University Medical Center. This study was supported by grants from the National Institutes of Health (P01 CA74175) and NeoPharm, Inc.

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Correspondence to Usha Kasid.

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Keywords

  • SCC-S2
  • subcellular localization
  • cell growth regulation in vitro and in vivo

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