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Molecular Biology and Genetics |
B Activity and PC-3 Human Prostate Cancer Cell Invasion1
Department of Pathology, Northwestern University, The Feinberg School of Medicine, Chicago, Illinois 60611 [J. C. H., P. F. L.], and Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 [J. B., S. K., A. K-B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NF-B) expression and activity were necessary for the highly invasive phenotype. In the current study, increased RhoA expression was found in the PC-3 highly invasive cells as compared with the PC-3 low invasive cells through cDNA array and Western blot analyses. Similarly, RhoA activity, as measured by the Rhotekin binding assay, was elevated in the PC-3 highly invasive cells. Transfection of these highly invasive cells with dominant negative RhoA N19 or treatment with 1.0 µg/ml RhoA inhibitor C3 exoenzyme demonstrated that RhoA activity was necessary for both NF-B activity and cellular invasion of a Matrigel reconstituted basement membrane. Furthermore, stable transfection of the PC-3 highly invasive cells with constitutively active RhoA Q63L resulted in activation of NF-B activity and Matrigel invasion, effects reversed by treatment of the cells with C3 exoenzyme. RhoA was also shown to act through the motility component of the invasion process. RhoA activity was therefore both necessary and sufficient for the elevated NF-B, invasion, and motility activities of the PC-3 highly invasive cells. These findings suggest molecular targets to control cancer cell invasion and aid in the development of definitive tools for predicting the invasive and metastatic potential of cancer cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cDNA array analysis indicated that PC-3 highly invasive cells have constitutively increased NF-B3
and RhoA expression compared with PC-3 low invasive cells. Such elevated NF-B activity has also been found in multiple aggressive cells lines including those of breast, squamous cell, and lung alveolar carcinomas (4, 5, 6)
. Furthermore, expression of dominant negative IB
S32/36A in the PC-3 highly invasive cells resulted in inhibition of both NF-B activity and invasion, indicating that increased NF-B activity is necessary for invasion of these cells (7)
. Similarly, inhibition of NF-B activity in the metastatic prostate cancer cell line PC-3M decreased invasion through Matrigel and metastasis upon injection into the prostate gland of nude mice (8)
.
The next step is to understand the role of RhoA in the highly invasive phenotype of PC-3 prostate cancer cells. RhoGTPases, including RhoA, are members of the Ras superfamily of monomeric 20–30 kDa GTP-binding proteins that act as molecular switches by alternating between active GTP-bound and inactive GDP-bound forms (9)
. This family of RhoGTPases is large, and its prototypic and most well-studied members are RhoA, Cdc42, and Rac1 (10)
. Besides their well-established roles in cytoskeletal regulation during processes such as cytokinesis (11, 12, 13, 14)
, smooth muscle contraction (11
, 15
, 16)
, and neurite retraction and rounding of neuronal cells (11
, 17
, 18)
, members of this family may also have potentially important roles in the invasive and metastatic behavior manifested by some cancers (19
, 20)
. The overexpression of one of these RhoGTPases, RhoA, occurs in some malignant tumors (21)
, and manipulation of the level of RhoA expressed in various transformed cell lines has been found to affect their level of aggressiveness (22, 23, 24, 25, 26, 27)
. RhoA can also affect gene expression through activation of transcription factors such as serum response factor (SRF) and NF-B (28)
and has been known to increase the expression of NF-B-dependent genes important for invasion (29, 30, 31, 32, 33, 34, 35, 36)
. We therefore tested the hypothesis that the highly invasive phenotype of the PC-3 cells was mediated, at least in part, through a highly active RhoA-NF-B pathway. Our findings indicate that RhoA activity was both necessary and sufficient for increased NF-B activity and invasion of PC-3 prostate cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transwell Invasion and Motility Assays.
The Transwell invasion assay has been described previously (38)
, with minor modifications (7)
. Briefly, 50,000 [3H]thymidine-labeled PC-3 cells were added to Matrigel-coated, porous upper chamber inserts in Transwell chamber plates (Costar, Corning, NY). The cells were then allowed to invade for 72 h without the addition of chemoattractant and then quantitated. The motility assay is a modified version of the Transwell invasion assay in which Matrigel was not added to the upper chamber inserts.
Transfection and NF-B Activity Assays (Luciferase Reporter Assay and EMSA).
Transfection of PC-3 cells, as described previously (7)
, occurred through the mixture of expression plasmids diluted in serum-free medium with LipofectAMINE (Life Technologies, Inc.). After a 30-min incubation at room temperature, the DNA-LipofectAMINE mixture was added to a cell monolayer for 5 h and then replaced with serum-containing medium and incubated for an additional 48 h at 37°C to allow sufficient time for gene expression. Transfection efficiency was determined through transfection of the cells with an EGFP reporter plasmid with subsequent counting of the fluorescent cells out of the total cell population using a Nikon Eclipse TE200 fluorescence microscope.
To assay the level of NF-B-dependent transcriptional activity in the PC-3 cells, the luciferase reporter assay was used as described previously (7)
. Briefly, this involved cotransfection of the cells with the pNF-B-Luc reporter plasmid (Clontech) and the pSV-ß-galactosidase control vector (Promega, Madison, WI). Simultaneously, the PC-3 cells were transfected with plasmids expressing dominant negative RhoA N19 (cDNA was a gift of Juan Carlos Lacal; Instituto de Investigaciones Biomedicas, Madrid, Spain; Ref. 31
) or its control vector, pcDNA3 (Invitrogen Life Technologies, Inc., Carlsbad, CA), constitutively active pEGFP-C1-RhoA Q63L expression plasmid (a gift of Joe Barbieri; Ref. 37
) or its control vector, pEGFP-C1 (Clontech). All luciferase results were normalized for ß-galactosidase activity.
The EMSA has been described previously (7)
and was used to assay NF-B activity at time periods shorter than the 48 h required after transfection for optimal gene expression in the luciferase reporter assay. The EMSA was used to study NF-B activity after treatment of the PC-3 cells with C3 exoenzyme (List Biological Laboratories, Inc., Campbell, CA), a specific inhibitor of RhoA (39, 40, 41)
.
Western Blot.
The Western blot procedure has been described previously, with minor modifications (7)
. The following antibodies were used: (a) 1:1000 dilution of anti-RhoA 119 (Santa Cruz Biotechnology, Santa Cruz, CA) with a 1:2000 dilution of HRP-linked antirabbit IgG as the secondary antibody; (b) 1:500 dilution of anti-Cdc42 (Santa Cruz Biotechnology) with a 1:2000 dilution of HRP-linked antirabbit IgG as the secondary antibody; and (c) a 1:1000 dilution of anti-Rac1 (Transduction Laboratories, Mississauga, Ontario, Canada) with a 1:2000 dilution of HRP-linked antimouse IgG as the secondary antibody.
RhoA (Rhotekin) Activity Assay.
RhoA activity was determined by measurement of RhoA-GTP binding to glutathione S-transferase-Rhotekin in a pull-down assay. This procedure has been described previously, with minor modifications (42)
. Briefly, PC-3 cells were exposed to lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 0.5% NP40, 1 µl/ml aprotinin (Roche Applied Science, Indianapolis, IN), 1 µl/ml leupeptin (Roche Applied Science), 1 µl/ml phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO), and 0.5 µl/ml DTT], and the cellular protein was harvested by scraping with a rubber policeman. Lysates were centrifuged at 4°C at 14,000 x g for 5 min to remove particulate material, and the protein concentrations were determined by the Bradford assay. Twenty-five µg of each protein extract were incubated, rotating at 4°C for 1 h, with an equal volume of pGEX-2T Rhotekin-RBD (RhoA-binding domain of the RhoA effector Rhotekin, a gift of Keith Burridge; The University of North Carolina at Chapel Hill, Chapel Hill, NC) bound to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotechnology, Uppsala, Sweden). The samples were then washed two times with lysis buffer, and RhoA protein was detected by Western blotting (see above).
Cell Proliferation and Viability Assays.
The Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR) was used to measure cell proliferation as described previously (7)
. Determination of cell viability was made using the trypan blue exclusion technique.
Statistics.
Statistical analysis was performed using InStat statistical software (GraphPad Software, Inc., San Diego, CA). For the invasion and MTT assay, the Student’s t test was used for comparisons. A nonparametric Wilcoxon’s paired test was used when analyzing data from the luciferase assay, which needed to be adjusted for transfection efficiency. Differences were considered significant when P was <0.05. The results are presented as mean ± SE.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In contrast, the expression levels of the related RhoGTPases Rac1 and Cdc42 were not significantly different between the invasion variant cell lines by Western blot (Fig. 1A)
or cDNA array analyses. The activity of RhoA was also shown to be increased >2-fold in the highly invasive cells compared with PC-3 low invasive cells (Fig. 1B)
. Thus, RhoA expression and activity were specifically increased in the highly invasive cells.
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B Activity by Inhibition of RhoA Activity.B and invasion activities shown by PC-3 highly invasive cells, the strategy was to inhibit RhoA activity and then test these parameters. PC-3 highly invasive cells were therefore transiently transfected with a vector expressing dominant negative RhoA N19, which reduced invasion from 4.4 ± 0.5% to 2.6 ± 0.25% invasion of control vector-transfected cells (P = 0.0017; Fig. 2A
). It is of note that transfection alone reduced the invasion of PC-3 cells, necessitating the comparison of cells transfected with RhoA N19 with control vector-transfected cells. In addition, we determined that PC-3 cells transfected under identical conditions with an EGFP reporter plasmid had a transfection efficiency of 41.4 ± 2.0%.
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B activity by 66 ± 4% (P < 0.0001; Fig. 2B
). The viability of the PC-3 highly invasive cells transfected with RhoA N19 was 95.0 ± 1.0%, which was not significantly different from the 95.5 ± 0.8% for the control vector-transfected cells. Similarly, PC-3 highly invasive cells transfected with RhoA N19 did not show a significant difference in viable cell number by MTT assay (P = 0.0002). Thus, the inhibition of NF-B activity and invasion caused by RhoA N19 were not due to an alteration in cell viability or proliferation.
To substantiate these effects of RhoA activity suppression, PC-3 highly invasive cells were treated with C3 exoenzyme, a specific biochemical inhibitor of RhoA (39, 40, 41)
. C3 exoenzyme (1.0 µg/ml) significantly reduced invasion from the 12.6 ± 0.6% invasion of untreated control cells to 6.4 ± 0.7% without significantly affecting cell viability (P < 0.0001; Fig. 3A
). In addition, the motility of PC-3 highly invasive cells was very significantly reduced from 9.9 ± 0.5% motility of untreated control cells to 3.5 ± 0.5% motility of C3 (1 µg/ml)-treated cells (P < 0.0001; Fig. 3B
).
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B activity was determined using EMSAs. EMSAs were used because the time periods of treatment were shorter than the 48 h required posttransfection for optimal gene expression in the luciferase reporter assay used previously. As seen before (7)
, untreated PC-3 highly invasive cells showed a 3.6-fold increase in specific NF-B DNA binding activity compared with untreated PC-3 low invasive cells (data not shown). Treatment of the PC-3 highly invasive cells with 1.0 µg/ml C3 exoenzyme resulted in a 37% inhibition of NF-B DNA binding activity within 6 h as compared with untreated PC-3 highly invasive cells (Fig. 3C)
. The NF-B complex specificity was determined by competition with mutant or wild-type oligonucleotides (Fig. 3C)
and by supershift assays (7)
.
The Rhotekin assay was used to confirm that C3 exoenzyme treatment of PC-3 highly invasive cells inhibited RhoA activity, showing a 32% decrease after 2 h and a 65% reduction after 48 h (Fig. 3D)
. In contrast, there was no significant change in the invasion activity of C3 exoenzyme-treated PC-3 low invasive cells (data not shown). Thus, both genetic and biochemical inhibition of RhoA activity blocked invasion and NF-B activity in PC-3 highly invasive cells, demonstrating the requirement of RhoA activity for these effects.
Elevation of Invasion and NF-B Activities in Stably Transfected Cells Expressing Constitutively Active RhoA.
To determine whether RhoA was sufficient to cause an increase in invasion activity, PC-3 highly invasive cells were transiently transfected with pEGFP-C1-RhoA Q63L, a plasmid expressing constitutively active RhoA fused to EGFP. Whereas this resulted in increased RhoA binding in the Rhotekin assay (Fig. 4A)
, there was no significant change in invasion compared with control vector-transfected cells (Fig. 4B)
. The transfection efficiency was also determined with this plasmid, and it ranged from 43% to 55% over the 72-h time period used in the invasion assay. As a control, MTT assays were performed, which showed no significant difference in viable cell number between PC-3 highly invasive cells transfected with pEGFP-C1-RhoA Q63L and those transfected with the control plasmid (P = 0.5374). Transient transfection of PC-3 low invasive cells with pEGFP-C1-RhoA Q63L also did not cause a significant change in NF-B activity or invasion (data not shown).
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). Stable RhoA Q63L cells also showed a parallel significant 2.3 ± 0.3-fold increase in NF-B activity compared with stable pEGFP-C1 control cells (P = 0.0005; Fig. 5B
). Although the total RhoA protein was seen in both stable cell lines, indicating a functioning assay, expression of the constitutively active EGFP-RhoA Q63L fusion protein was below the level of detection with anti-Rho antibodies (data not shown). However, a Western blot of the stable cells probed with an anti-EGFP antibody showed EGFP in stable pEGFP-C1 control vector-transfected cells, whereas the EGFP-RhoA Q63L fusion protein was detected in the stable RhoA Q63L cells (Fig. 5C)
. It is of note that these stable cell lines were not clonally selected.
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, there was again a significant increase in the invasion activity from 6.6 ± 1.0% invasion of the untreated stable pEGFP-C1 control cells to 11.1 ± 1.2% of the untreated stable RhoA Q63L cells (Fig. 6A)
. Exposure of the cells to C3 exoenzyme significantly inhibited invasion of the stable RhoA Q63L cells from 11.1 ± 1.2% for untreated control cells down to 4.9 ± 0.5% for C3-treated cells (P = 0.0037) but did not affect stable pEGFP-C1 control cell invasion (Fig. 6A)
. These results support a role for elevated RhoA activity in the increased invasion shown by stable RhoA Q63L cells compared with stable pEGFP-C1 control cells.
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). C3 exoenzyme also inhibited the motility of stable pEGFP-C1 control cells, but to a lesser degree than the stable RhoA Q63L cells (Fig. 6B)
. Thus, similar to the observations in PC-3 highly invasive cells, in stable RhoA Q63L cells, motility appears to be a major component of invasion affected by the RhoA-induced increase in invasion. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activities and the invasiveness of PC-3 human prostate cancer cells through comparison of high and low invasion variants. Previously, as compared with the low invasion variant, PC-3 highly invasive cells were shown to exhibit increased activity of NF-B (7)
, a transcription factor known to regulate genes that control the immune response, growth, and apoptosis and, more recently, genes important for invasion and metastasis including urokinase-like plasminogen activator, MMP-9, and tissue factor (4, 5, 6
, 8
, 34, 35, 36)
. Similarly, compared with PC-3 low invasive cells, the expression and activity of RhoA were constitutively increased in the highly invasive prostate cancer cells (Fig. 1, A and B)
. This is in agreement with findings of elevated RhoA levels in aggressive colon, lung, and breast tumors (21)
.
Through transient expression of dominant negative RhoA N19, RhoA activity was found to be necessary for the increased invasion and NF-B activities of PC-3 highly invasive cells (Fig. 2, A and B)
. Although such manipulation of RhoA activity has been shown to affect the level of aggressiveness of other transformed cell lines (22, 23, 24, 25, 26, 27)
, we revealed a previously unknown relationship by directly demonstrating a requirement of RhoA activity for both invasion and NF-B activity. The effects of RhoA inhibition were confirmed though exposure of the PC-3 highly invasive cells to C3 exoenzyme, a biochemical inhibitor of RhoA activity, which resulted in decreased RhoA, NF-B, motility, and invasion activities (Fig. 3, A–D)
.
Our finding that RhoA is necessary for increased NF-B, invasion, and motility activities is supported by a study of cerivastatin in breast cancer cells (43)
. Cerivastatin prevents the synthesis of cholesterol precursors including GGPP, an isoprenoid that causes prenylation of Rho, resulting in its translocation to the cell membrane and subsequent cell signaling. Exposure of the highly invasive and metastatic breast cancer cell line MDA-MB-231, which has constitutively elevated RhoA and NF-B activities, to cerivastatin inhibited cellular invasion and motility. This effect on invasion was attributed to inhibition of RhoA because the invasive phenotype was restored after addition of GGPP. NF-B activity was also decreased in a RhoA-dependent manner in the cells after cerivastatin treatment. This indirect look at RhoA through the negative action of cerivastatin on GGPP supports a role for RhoA and NF-B in breast cancer cell invasion. Furthermore, because these findings occurred in breast cancer cells, whereas our direct results showing the requirement for RhoA were found in prostate cancer cells, this suggests that up-regulation of a RhoA-NF-B pathway is possibly a general mechanism by which multiple types of aggressive tumor cells invade.
Because the invasion process is comprised of multiple steps including cellular movement and expression of membrane-degrading enzymes such as the matrix metalloproteinases (44)
, the effect of C3-induced inhibition of RhoA in the PC-3 highly invasive cells on these invasion components was determined. Using zymogen gels,4
PC-3 highly invasive cells were found to have the same expression level of MMP-2 and MMP-9 as the PC-3 low invasive cells, thus suggesting that the highly invasive phenotype did not result from up-regulation of these matrix-degrading enzymes. However, the motility of PC-3 highly invasive cells was very significantly reduced by exposure of the cells to C3, indicating that RhoA affects invasion through the motility component (Fig. 3B)
.
Cell motility is regulated by RhoA, Rac1, and Cdc42 RhoGTPases through stimulation of actin cytoskeletal rearrangements. RhoA promotes the development of focal adhesions and stress fibers, whereas Rac1 induces the formation of lamellipodia, and Cdc42 causes filopodia development and establishment of cellular polarity (45, 46, 47, 48, 49, 50)
. Although RhoA has been shown here to have elevated expression and be integrally involved in the motility and invasion of PC-3 highly invasive cells, surprisingly, the expression levels of Rac1 or Cdc42 did not vary between the two invasion variant cell lines (Fig. 1)
. Additional studies will be performed to look for differences in their activity levels.
The question of RhoA sufficiency for the invasive phenotype was addressed by transfecting pEGFP-C1-RhoA Q63L, a plasmid expressing constitutively active RhoA fused to EGFP, into the highly invasive cells. Transient transfection of the cells with this plasmid resulted in increased RhoA activity in the Rhotekin assay (Fig. 4A)
, but no significant change in invasion compared with control vector-transfected cells occurred (Fig. 4B)
. In contrast, when cells stably expressing pEGFP-C1-RhoA Q63L were compared with control vector-transfected stable cells, they showed increased invasion and NF-B activities (Fig. 5, A and B)
. Treatment of these stable RhoA Q63L cells with C3 exoenzyme resulted in significant inhibition of invasion, providing evidence that the increased invasion shown by the stable cells was due specifically to RhoA activity (Fig. 6A)
. Furthermore, the expression of the EGFP-RhoA Q63L fusion protein in these stable RhoA Q63L cells was demonstrated by Western blot probed with anti-EGFP (Fig. 5C)
. It is of note that the fusion protein in the stable cells was at the limit of detection with anti-RhoA antibody, providing the possibility that the EGFP-RhoA Q63L fusion protein was present at a lower level in stably transfected cells as compared with transiently transfected cells. Nevertheless, the stable RhoA Q63L transfection-induced level of RhoA activity was sufficient to increase invasion and NF-B activity in the highly invasive cells.
Additionally, similar to the observations in PC-3 highly invasive cells, motility seems to be a major component of invasion affected by RhoA in the stable RhoA Q63L cells (Fig. 6B)
. This agrees with several studies in which RhoA was required for motility, whereas highly elevated RhoA activity inhibited movement (27
, 50, 51, 52)
. Similar RhoA activity dosage effects have been described in fibroblasts, leading to the proposal that high levels of RhoA activity may inhibit motility and therefore invasion by causing uncontrolled adhesion and disrupting cell polarization (53)
.
In summary, our experiments support a model of the PC-3 highly invasive cells in which constitutively increased RhoA activity leads to elevated NF-B transcriptional activity and invasion. RhoA appears to be both necessary and sufficient for this pathway, and additional studies currently underway will determine the clinical applications of these findings.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Department of Defense Grant DAMD17-02-1-0162. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Northwestern University, The Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611. Phone: (312) 926-8483; Fax: (312) 503-8249; E-mail: p-lindholm{at}northwestern.edu
3 The abbreviations used are: NF-B, nuclear factor B; EMSA, electrophoretic mobility shift assay; EGFP enhanced green fluorescent protein; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MMP, matrix metalloproteinase; GGPP, geranylgeranyl PPi.
4 Dr. Sally Twining, personal communication.
Received 10/ 3/02. Accepted 12/26/02.
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REFERENCES |
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3ß1 and 6ß4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane. Clin. Exp. Metastasis, 11: 391-400, 1993.[Medline]
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B and C/EBP transcription factors. J. Biol. Chem., 274: 8506-8515, 1999.B activation and IL-1ß gene expression in cultured human epithelial cells. J. Immunol., 160: 3038-3045, 1998.B by Rho, CDC42, and Rac-1 proteins. Genes Dev., 11: 463-475, 1997.B by genotoxic stress. Exp. Cell Res., 264: 244-249, 2001.[Medline]
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