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Originally published In Press as doi:10.1074/jbc.M604636200 on June 5, 2006

J. Biol. Chem., Vol. 281, Issue 31, 22100-22107, August 4, 2006
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Up-regulation of Skp2 after Prostate Cancer Cell Adhesion to Basement Membranes Results in BRCA2 Degradation and Cell Proliferation*

Loredana Moro{ddagger}1, Arnaldo A. Arbini§, Ersilia Marra{ddagger}, and Margherita Greco{ddagger}

From the {ddagger}Institute of Biomembranes and Bioenergetics, National Research Council, Bari 70126, Italy and the §Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication, May 15, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Basement membranes (BM)2 are thin layers of specialized extracellular matrix (ECM) that surround and closely associate with epithelial and endothelial cells, muscle fibers, and nerves. They consist mostly of collagen type IV (COL4) admixed with laminins (LN), nidogens, and the heparan sulfate proteoglycan perlecan (PLN) and may contain small amounts of fibronectin (FN) (1, 2). Although the BM structural role in defining tissue architecture and compartmentalization has long been recognized, its dynamic role in the modulation of cell behavior has only recently been documented (1).

Aberrant cancer cell interactions with BM proteins play a crucial role in the biology of metastasis (3). Cancer cells must be able to coordinately decrease cell-cell interactions and increase cell adhesion to an adjacent BM in order to become motile, which along with the capacity of degrading/remodeling a BM directly relates to their metastatic potential (4). This cell behavior is accompanied by changes in the expression and/or usage of various adhesion receptors, including integrins (5). Integrins are transmembrane adhesion receptors for ECM proteins that not only provide physical anchoring cell support but also play a pivotal role in triggering intracellular signaling in response to environmental changes through interactions with molecules such as growth factor receptor tyrosine kinases (6, 7), MAPK/ERK 1/2 (8, 9), and PI 3-kinase/AKT (10, 11). These various interactions help in modulating the expression of genes exerting stringent control upon cell survival, motility, and cell proliferation (12).

Intracellular protein degradation via the ubiquitin-proteasome pathway is a prime pathway through which cells normally regulate processes involved in cell growth and proliferation (13, 14). There is evidence that a number of growth inhibitory molecules and tumor suppressor proteins, such as p53, p21, p27, p130, the beta1C integrin, and FOXO1, are preferentially degraded by the ubiquitin-proteasome system in carcinoma cells (14-17). Furthermore, E3 ubiquitin ligase family members Skp2 and Mdm2 have been shown to play a role in prostate cancer development and progression (18-20). In a previous report, we provided evidence for a novel pathological mechanism whereby prostate carcinoma cell adhesion to collagen type I (COL1), a major ECM protein at osseous metastatic sites, promotes cancer cell proliferation through depletion of BRCA2 protein, the product of a tumor suppressor gene whose inactivation accounts for an increased risk in cancer development (21-23). This newly described effect resulted from beta1 integrin-dependent activation of the PI 3-kinase pathway, which promoted BRCA2 ubiquitination and degradation in the proteasome (24).

In this study, we extended our investigations to elucidate the mechanisms by which beta1 integrin signaling in prostate cancer cells resulted in BRCA2 protein degradation in the proteasome. We also provide evidence demonstrating that the BRCA2-associated trophic effect is not restricted to the osseous environment but is quite active in mediating cancer cell proliferation after interaction with BM proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—PNT1A cells (a human prostate normal cell line established by immortalization of normal adult prostate epithelial cells) and PC-3 cells (a human prostate carcinoma cell line derived from a bone metastasis) were kept in culture as described previously (16, 24).

Cell Adhesion—Cell adhesion assays to ECM proteins were carried out using 96-well tissue culture plates as described previously (24). Plates were precoated with different concentrations of FN (3 µg/ml; Sigma), LN (10 µg/ml; Invitrogen), PLN (10 µg/ml; Sigma), or COL4 (10 µg/ml; Sigma) for 16 h at 4 °C. Coating with 10 µg/ml bovine serum albumin (Sigma) served as negative control.

Adhesion to the BM matrix Matrigel (Sigma) was tested in 96-well plates coated with 50 µl/well of a 1:3 dilution in RPMI medium (Invitrogen) before cell plating. Cells were starved in serum-free methionine/cysteine-deficient RPMI 1640 (Sigma) for 45 min at 37 °C before labeling with 100 µCi/ml 35S protein-labeling mix (Amersham Biosciences) in 1 ml of methionine/cysteine-free RPMI medium containing 5% fetal bovine serum. After 24 h, 100 µlofa0.2 x 106 cell suspension were allowed to adhere for 1 h onto Matrigel or bovine serum albumin (10 µg/ml) at 37 °C and were washed three times. Adherent cells were lysed in 100 µl of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA containing 1% (v/v) Triton X-100 in phosphate-buffered saline. Radioactivity was measured in a scintillation counter (Beckman Instruments).

Inhibition assays were performed by incubating cells for 1 h on ice in the presence of either P4C10, a monoclonal antibody to human beta1 integrin (ascites 1:200; Chemicon, Temecula, CA), or the monoclonal antibody 1C10 against a vascular endothelial surface protein (ascites 1:200; Invitrogen) used as a negative control. Triplicate observations were recorded for each experiment.

Immunoblotting Analysis and Immunoprecipitation—Cells were grown either onto FN (3 µg/ml), LN (10 µg/ml), PLN (10 µg/ml), or COL4 (10 µg/ml) and lysed, and protein extracts were analyzed by immunoblotting as described previously (24). Where indicated, cells were pretreated for 1 h with either P4C10 or 1C10, or the PI 3-kinase inhibitors wortmannin (0.1 µM; Sigma), LY294002 (10 µM; Calbiochem), or solvent alone (Me2SO), or added with the proteasome inhibitor MG132 (10 µM). The following antibodies were used: 1 µg/ml anti-BRCA2 polyclonal antibody (H-300; Santa Cruz Biotechnology, Santa Cruz, CA), 10 µg/ml monoclonal antibody to beta-tubulin (Sigma), 1 µg/ml polyclonal antibody to Skp2 (H-435; Santa Cruz Biotechnology), 2 µg/ml monoclonal antibody to Mdm2 (D-12; Santa Cruz Biotechnology), 1 µg/ml anti-phospho-AKT-Ser-473 polyclonal antibody (Santa Cruz Biotechnology), 1 µg/ml anti-AKT 1/2 polyclonal antibody (H-136; Santa Cruz Biotechnology), 0.2 µg/ml anti-phospho-ERK monoclonal antibody (E-4; Santa Cruz Biotechnology), 0.2 µg/ml anti-ERK2 polyclonal antibody (C-14; Santa Cruz Biotechnology), or 1:1000 dilution of anti-p85{alpha} rabbit antiserum (Sigma).

Analysis of BRCA2 ubiquitination was performed as described previously (24). To analyze BRCA2 association with Skp2, cell extracts were precleared and incubated overnight with 2 µg of polyclonal antibody to BRCA2. Immunocomplexes were recovered with protein A-Sepharose (Sigma), washed five times with phosphate-buffered saline containing 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate and were subjected to 10% SDS-PAGE under reducing conditions followed by transfer to polyvinylidene difluoride membranes. Filters were immunoblotted using 1 µg/ml monoclonal antibody to Skp2 (Zymed Laboratories Inc., San Francisco, CA) or 2 µg/ml monoclonal antibody to BRCA2 (clone 5.23; Chemicon) following the manufacturers' instructions. Alternatively, whole cell extracts were immunoprecipitated with 2 µg of monoclonal antibody to Skp2 (Zymed Laboratories Inc.) and separated by 6% SDS-PAGE, and filters were immunoblotted using 1 µg/ml anti-BRCA2 polyclonal antibody or 1 µg/ml polyclonal antibody to Skp2.

Transient Transfections and [3H]Thymidine Incorporation— Transient transfections with wild-type BRCA2 cDNA (a kind gift from Dr. M. C. Hung, University of Texas M. D. Anderson Cancer Center, Houston), {Delta}p85, a dominant negative form of PI 3-kinase (generously provided by Dr. R. Freeman, University of Rochester, Rochester, NY), or empty vector (pcDNA3; Invitrogen) were performed as described previously (24). A pool of siRNAs for human Skp2, Mdm2, and nonspecific siRNAs was purchased from Santa Cruz Biotechnology and used for transient transfections according to the manufacturer's instructions. Thymidine incorporation assays were performed in 96-well plates as described previously (24).

Statistical Analysis—Data are reported as the mean ± S.E. Statistical analysis was performed by the Student's t test. All experiments were repeated at least twice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Prostate Normal and Carcinoma Cells Adhere Differently to BM—We investigated the adhesive properties of PNT1A and PC-3 cells to LN, PLN, and COL4, major components of BM, and to FN, a widely expressed extracellular matrix protein (25) enriched at the prostatic stroma but a minor component in BM. As shown in Fig. 1A, PNT1A cells adhered efficiently to FN, to a lesser extent to LN and PLN (~60% of FN levels), and showed no adhesion to COL4. Highly metastatic PC-3 cells exhibited a reversed affinity pattern, with partial loss of adhesion to FN and newly gained adhesion capabilities to BM proteins. Because cell adhesion to FN, LN, COL4, and PLN (26) is mediated by integrin receptors that share a common beta1 subunit ({alpha}5beta1 to FN, {alpha}6beta1 to LN, {alpha}1beta1, {alpha}2beta1, and {alpha}3beta1 to COL4, {alpha}beta1 to PLN), we tested the effect of the beta1 integrin-blocking antibody P4C10 on cell adhesion. As shown in Fig. 1B, adhesion to FN was reduced by 42 ± 6% (p < 0.002) and 39 ± 7% (p < 0.001) in PNT1A and PC-3 cells, respectively. Adhesion to LN was inhibited by ~25% in PNT1A and ~75% in PC-3 cells, adhesion to PLN was almost completely inhibited in both cell types, and onto COL4 PC-3 cell adhesion was almost completely inhibited (90 ± 7%, p < 0.0001).


Figure 1
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  		FIGURE 1.
Prostate normal and carcinoma cells adhere differently to BM. A, 1.5 x 105 PNT1A or PC-3 cells were allowed to adhere to bovine serum albumin (BSA) (10 µg/ml), FN (3 µg/ml), LN (10 µg/ml), PLN (10 µg/ml), or COL4 (10 µg/ml) at 37 °C for 1 h and were washed and stained with crystal violet. Cell adhesion was quantified by measuring the absorbance at 630 nm in an enzyme-linked immunosorbent assay reader. B, 1.5 x 105 PNT1A or PC-3 cells were incubated for 1 h on ice in the presence of the beta1 integrin-blocking antibody P4C10 or 1C10 as control and allowed to adhere to bovine serum albumin (BSA), FN, LN, PLN, or COL4 as described in panel A. C, 2 x 105 35S-labeled PNT1A or PC-3 cells were incubated for 1 h on iceinthe presence or absence of P4C10 or 1C10 before being allowed to adhere to Matrigel (~0.2 mg of protein/well) for 1 h at 37°C. Following three phosphate-buffered saline washes, cells were lysed and the radioactivity was measured. Data are expressed as mean ± S.E. of triplicate wells. A representative experiment of three is shown.

 
To investigate whether these findings could be reproduced onto naturally occurring BM layers, we tested the adhesive properties of normal and cancer prostate cells to Matrigel, a reconstituted BM preparation (27). As shown in Fig. 1C, whereas PC-3 cells adhered efficiently to Matrigel in a manner that was significantly inhibited by the anti-beta1 integrin-blocking antibody (~70% inhibition, p < 0.004), normal prostate cell adhesion was nil in the time period considered.

Adhesion of PC-3 Cells to BM Proteins Decreases BRCA2 Protein Levels in a beta1 Integrin-dependent Manner—We asked whether PC-3 cell adhesion to BM proteins exercised a modulatory effect upon BRCA2 expression. To this effect, PNT1A and PC-3 cells were grown onto plates coated with FN, LN, PLN, COL4, or Matrigel and BRCA2 levels were assessed. As depicted in Fig. 2A, whereas PNT1A cells transiently increased BRCA2 protein after adhesion to BM proteins (2.2 ± 0.3-fold onto FN after 2.5 h, p < 0.002), PC-3 cells exhibited almost complete (PLN) or complete (LN, COL4, Matrigel) loss of detectable BRCA2 after adhesion for 6 h. The strongest down-regulatory effect was observed onto COL4, which caused BRCA2 protein levels to decrease after only 2.5 h by 68 ± 4% (p < 0.01). BRCA2 protein levels did not recover after 12 h of cell adhesion (data not shown). The beta1 integrin-blocking antibody P4C10 partly rescued BRCA2 protein to 45 ± 6% (p < 0.001) after 6 h onto COL4 (Fig. 2B) as well as onto other BM proteins (data not shown). As COL4 is the most abundant component of the BM, most of the subsequent experiments were performed onto this BM protein.

BRCA2 Protein Depletion after Cancer Cell Adhesion to BM Increases DNA Synthesis—To investigate whether the signaling cascade initiated by BM proteins had any effect on cell proliferation, we measured DNA incorporation of [3H]thymidine in PNT1A and PC-3 cells onto various substrates (Fig. 3A). Whereas PNT1A cell proliferation showed no variations irrespective of the adhesive substrate, adhesion to COL4 enhanced PC-3 cell proliferation compared with plastic by 291 ± 24% (p < 0.001). A somewhat less strong proliferative response was elicited upon adhesion onto LN (~213%) and PLN (~175%). No significant change was detected onto FN (data not shown). Preincubation of PC-3 cells with P4C10 inhibited the response to COL4 by ~31% and to LN and PLN by ~35% (data not shown). These results were further replicated and confirmed onto native BM tissue. As shown in Fig. 3B, adhesion to Matrigel enhanced cell proliferation by 509 ± 27% and P4C10 inhibited the response by 32 ± 5% (p < 0.0002). The increase in DNA synthesis after adhesion to BM proteins could be reversed by transfecting PC-3 cells with wild-type BRCA2 cDNA (Fig. 3C). In these experiments, PC-3 cells were transiently transfected with BRCA2 cDNA or empty vector for 36 h, after which we measured BRCA2 protein levels (upper panel) and [3H]thymidine incorporation upon 12 h of cell adhesion to COL4 in the presence or absence of the proteasome inhibitor MG132 (lower panel). After transfection, BRCA2 protein levels increased 2.3-fold compared with mock-transfected cells. Upon COL4 adhesion, BRCA2 decreased by 44 ± 8% (p < 0.03) in transfected cells, but this reduction had no effect in [3H]thymidine incorporation, which remained at basal levels throughout the experiment. On the contrary, mock-transfected cells exhibited complete disappearance of BRCA2 protein upon adhesion to COL4, which resulted in a burst in [3H]thymidine incorporation. This trophic effect could be reversed with the proteasome inhibitor MG132, which decreased [3H]thymidine incorporation by 94% (p < 0.003). Treatment of BRCA2-transfected cells with MG132 resulted in BRCA2 accumulation to 245 ± 29% (p < 0.001).

Skp2 Promotes BRCA2 Protein Depletion in PC-3 Cells Adherent to BM—Because it is known that F-box proteins Skp2 and Mdm2 mediate the ubiquitin-dependent degradation of several negative regulators of cell proliferation in cancer (17, 28, 29), we hypothesized that they were likely candidates to be involved in BRCA2 ubiquitination and proteasomal degradation in prostate cancer cells. We first investigated whether PC-3 cell adhesion to COL4 had any effect upon Skp2 or Mdm2 protein levels. As depicted in Fig. 4A, PC-3 cell adhesion to COL4 triggered Skp2 and Mdm2 protein expression levels by 281 and 184% at 2.5 h and 393 and 203% at 6 h, respectively. This upregulatory effect was significantly inhibited by a beta1 integrin-blocking antibody for Skp2 but not Mdm2. We then proceeded to investigate whether manipulation of Skp2 or Mdm2 levels had an effect upon BRCA2 protein levels and DNA synthesis. As shown in Fig. 4B, Skp2 knock down by siRNAs resulted in Skp2 protein cell depletion (upper panel) and concomitant rescue of BRCA2 protein levels after adhesion to COL4 (lower panel). Furthermore, these newly induced changes blunted much of the new DNA synthesis upon PC-3 cell adhesion to COL4 (Fig. 4C). BRCA2 protein depletion was independent from any increase in Mdm2 protein levels upon adhesion to COL4, and Mdm2 knock down by siRNAs did not rescue BRCA2 protein levels (data not shown).


Figure 2
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  		FIGURE 2.
PC-3 cell adhesion to BM proteins decreases BRCA2 protein levels in a beta1 integrin-dependent manner. A, PNT1A and PC-3 cells were allowed to adhere to FN (3 µg/ml), LN (10 µg/ml), PLN (10 µg/ml), COL4 (10µg/ml), or Matrigel at 37 °C for 0-6 h and were washed and processed for immunoblotting with anti-BRCA2 antibody. beta-Tubulin signals were used as loading controls. The blots are representative of three independent experiments. Bottom, BRCA2 protein levels were quantitated and reported as percentage of the amount of protein at 0 h, set to represent 100%. Data are expressed as mean ± S.E. of three independent experiments. B, PC-3 cells were incubated for 1 h on ice in the presence of the beta1 integrin-blocking antibody P4C10 or 1C10 as control, allowed to adhere to COL4 (10 µg/ml) at 37 °C for 0-6 h, and processed for immunoblotting. Membranes were probed with anti-BRCA2 antibody. beta-Tubulin signals were used as loading controls. The blots are representative of three independent experiments.

 
Skp2 Directly Interacts with BRCA2, Promoting Its Ubiquitination—Next we examined whether changes in Skp2 expression had any effect upon BRCA2 ubiquitination in PC-3 cells after COL4 adhesion. To this effect, cell extracts from PC-3 cells transfected with Skp2 siRNAs or mock transfectants were prepared at various time points and immunoprecipitated with anti-BRCA2 antibody, followed by Western blotting with an antibody against ubiquitin (Fig. 5A). Before adhesion (0 h), PC-3 cells showed minimal BRCA2 ubiquitination, as previously described (24). After 2.5 h of adhesion to COL4, cells transfected with nonspecific siRNAs exhibited steady increase in BRCA2 ubiquitination at a pace that mirrored the reduction in BRCA2 protein levels shown in Fig. 2A. In contrast, knock down of Skp2 expression resulted in a dramatic reduction in BRCA2 ubiquitination at all time points. This effect results from a direct interaction between the two proteins as demonstrated by coimmunoprecipitation assays. In the experiment shown in Fig. 5B, after COL4 adhesion amounts of Skp2-BRCA2 complex immunoprecipitated with anti-BRCA2 sharply increased from detectable levels at 0 h to maximal amounts at 2.5 h to subsequently decrease after 6 h, reflecting virtual BRCA2 protein depletion. This behavior mirrored the changes in Skp2 depicted in Fig. 4A. BRCA2 was also detected when the anti-Skp2 antibody was used for immunoprecipitation (data not shown).

PC-3 Cell Adhesion to BM Triggers PI 3-Kinase Activation in a beta1 Integrin-dependent Manner—We also investigated the involvement of the MAPK/ERK and the PI 3-kinase signaling pathways in modulating BRCA2 protein depletion upon cancer cell adhesion to BM. In Western blotting with anti-phospho-ERK, MAPK/ERK activity in PC-3 cells was nil at rest and did not increase after adhesion to COL4 (Fig. 6A). On the contrary, PI 3-kinase activity increased by 2-fold, remaining highly phosphorylated for as long as 6 h as measured by Western blotting with anti-phospho-Ser-473 AKT. This response was dependent on beta1 integrin as pretreatment with the blocking antibody P4C10 resulted in ~80% inhibition in AKT phosphorylation onto COL4 (Fig. 6B). AKT phosphorylation affected BRCA2 levels as demonstrated by inhibition of PI 3-kinase activity with wortmannin (Fig. 6C) or LY294002 (data not shown), which increased BRCA2 protein by 4.3-fold after 2.5 h of adhesion to COL4.

Degradation of BRCA2 by Skp2 Requires PI 3-Kinase—To investigate whether the increase in Skp2 protein levels leading to BRCA2 ubiquitination after PC-3 cell adhesion to COL4 results from aberrant PI 3-kinase activation, we transiently transfected PC-3 cells with a dominant negative ({Delta}p85) form of the PI 3-kinase before adhesion to COL4 and analyzed protein levels of BRCA2, Skp2, and Skp2-BRCA2 complex and [3H]thymidine incorporation. As depicted in Fig. 7, transfection with {Delta}p85 completely prevented BRCA2 protein degradation and Skp2 protein up-regulation (panel A) as well as the increase in Skp2-BRCA2 protein complex (panel B) and DNA synthesis (panel C) upon cell adhesion to COL4.


Figure 3
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  		FIGURE 3.
BRCA2 protein depletion after cancer cell adhesion to BM proteins increases DNA synthesis. A and B, PNT1A or PC-3 cells were preincubated for 1 h on iceinthe presence or absence of beta1 integrin-blocking antibody P4C10 or 1C10 as control and cultured onto PL, FN (3 µg/ml), COL4 (10 µg/ml), or Matrigel (~0.2 mg of protein/well) for 15 h. A pulse with 1 µCi/well of methyl-[3H]thymidine was applied for the last 3 h. After washing with cold medium, cells were solubilized and collected into scintillation vials for quantification of incorporated radioactivity. Data are expressed as mean ± S.E. of triplicate wells. A representative experiment of three is shown. C, PC-3 cells were transiently transfected with wild-type BRCA2 cDNA or empty vector (pcDNA3) for 36 h, after which BRCA2 protein expression was determined by immunoblotting before (Control) and after 12 h of adhesion to COL4 in the presence or absence of the proteasome inhibitor MG132 (10 µM). Bottom, PC-3 cells transiently transfected for 36 h with BRCA2 cDNA or vector alone were allowed to adhere onto plastic or COL4 in presence of MG132 or the solvent alone (-) and pulsed with methyl-[3H]thymidine as described in panels A and B. Data are expressed as mean ± S.E. of triplicate wells. A representative experiment of three is shown.

 


Figure 4
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  		FIGURE 4.
Skp2 promotes BRCA2 protein depletion in PC-3 cells adherent to BM. A, PC-3 cells were preincubated for 1 h on ice in the presence or absence of beta1 integrin-blocking antibody P4C10, allowed to attach to COL4 for the indicated times, and washed and processed for immunoblotting. Membranes were probed with anti-Skp2, anti-Mdm2, and beta-tubulin as loading control. The blots are representative of three independent experiments. Bottom, Skp2 and Mdm2 protein levels were quantitated and reported as percentage of the amount of protein at 0 h, set to represent 100%. Data are expressed as mean ± S.E. of three independent experiments. B, PC-3 cells were transfected with a pool of nonspecific siRNAs (-) or siRNAs for Skp2, and protein knockdown was determined by immunoblotting for Skp2 36 h after transfection. Bottom, PC-3 cells 36 h after transient transfection with Skp2 siRNAs were allowed to adhere to COL4 for the indicated times and processed for immunoblotting for BRCA2, Skp2, or beta-tubulin as loading control. C, PC-3 cells transiently transfected with interfering RNAs for Skp2 or nonspecific siRNAs (n.s. siRNA) were allowed to adhere to COL4 for 15 h and pulsed with methyl-[3H]thymidine for the last 3 h. Data are expressed as mean ± S.E. of triplicate wells. A representative experiment of two is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The ability of carcinoma in situ to overcome the surrounding epithelial BM and spread into the underlying stroma has long defined the progression to invasive carcinoma (30). In turn, for local and distant metastasis to occur, locally invasive carcinoma cells must be able to colocalize and eventually invade muscle, nerves, and blood vessels, all of which are separated from the adjacent stroma by continuous sheets of BM. Hence, a fundamentally abnormal interaction of carcinoma cells with BM proteins must be at the core of their metastatic capabilities. Our experimental paradigm confirmed that unlike normal PNT1A, highly invasive PC-3 carcinoma cells exhibited inappropriate adhesion onto the main components of the BM as well as on a reconstituted BM matrix system. On the other hand, the ECM glycoprotein FN enriched at the prostatic stroma and a minor component of BM was a poor adhesive substrate for neoplastic PC-3 cells when compared with normal PNT1A cells. This latter observation could be partly correlated with the lower expression of the FN receptor beta3 integrin in PC-33 when compared with PNT1A cells.


Figure 5
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  		FIGURE 5.
Skp2 directly interacts with BRCA2, promoting its ubiquitination. A, PC-3 cells were transfected with a pool of siRNAs for Skp2 or nonspecific siRNAs (-); 36 h after transfection cells were allowed to adhere to COL4 for the indicated times and lysed, and protein extracts were immunoprecipitated (IP) with a BRCA2 antibody. Immunoprecipitates were analyzed by immunoblotting (IB) with anti-ubiquitin and anti-BRCA2 antibody. Blots are representative of two independent experiments. B, PC-3 cells were allowed to adhere to COL4 for the indicated times and lysed, and protein extracts were immunoprecipitated with anti-BRCA2 antibody and immunoblotted with antibodies for Skp2 and BRCA2. Blots are representative of three independent experiments.

 


Figure 6
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  		FIGURE 6.
PC-3 cell adhesion to COL4 triggers PI 3-kinase activation in a beta1 integrin-dependent manner. A, PC-3 cells were allowed to attach to COL4 at 37 °C for 0-6 h, washed and lysed, and 100 µg of cell protein extracts were electrophoresed onto 10% SDS-polyacrylamide gel under reducing conditions. Membranes were probed with anti-phospho-Ser-473 AKT (P-AKT) or anti-phospho-ERK (P-ERK). Subsequently, membranes were stripped and blotted with anti-AKT 1/2 (AKT 1/2) or anti-ERK2 (ERK2) as control. The blots are representative of three independent experiments. B, PC-3 cells were preincubated with the beta1 integrin-blocking antibody P4C10 or 1C10 as control before plating onto COL4 and being processed as described in panel A. The blots are representative of two independent experiments. C, PC-3 cells were pretreated for 1 h at 37 °C with 0.1 µM wortmannin (WM), a PI 3-kinase inhibitor, or the solvent alone (Me2SO). Cells were then allowed to adhere to COL4 at 37 °C for 0 or 2.5 h and were washed, lysed, and processed for immunoblotting. Membranes were probed with anti-BRCA2 antibody and beta-tubulin as loading control. The blots are representative of two independent experiments. Bottom, BRCA2 protein levels were quantitated and reported as percentage of the amount of protein at 0 h, set to represent 100%. Data are expressed as mean ± S.E. of two independent experiments.

 
Another crucial finding is that after adhesion to isolated BM proteins, particularly to COL4, highly invasive carcinoma cells exhibit a burst in [3H]thymidine incorporation, a sensitive measurement of new DNA synthesis and cell proliferation. This trophic response seems to be elicited by beta1 integrin adhesion, as a similar phenomenon was previously demonstrated onto COL1, a ligand for beta1 integrin receptors (24). The proliferative response after adhesion to LN and PLN was slightly weaker than with COL4, and no effect was noticeable onto FN. When allowed to adhere onto the BM matrix Matrigel, prostate cancer cells exhibited a proliferation surge that was stronger than with isolated components, suggesting cooperation rather than competition among various proteins in activating proliferative signals.


Figure 7
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  		FIGURE 7.
Degradation of BRCA2 by Skp2 requires PI 3-kinase. A and B, PC-3 cells were mock transfected (pcDNA3) or received dominant negative PI 3-kinase p85 subunit ({Delta}p85). After 36 h, cells were allowed to adhere to COL4 at 37 °C for the indicated times, lysed, and processed for immunoblotting with antibodies against BRCA2, Skp2, or tubulin as loading control (A) or first immunoprecipitated (IP) with anti-BRCA2 and then immunoblotted (IB) with a monoclonal antibody against ubiquitin (B). The blots are representative of two independent experiments. At the bottom of panel A, BRCA2 and Skp2 protein levels were quantitated and reported as percentage of the amount of protein at 0 h, set to represent 100%. Data are expressed as mean ± S.E. of two independent experiments. C, PC-3 cells transiently transfected with dominant negative PI 3-kinase or vector alone were allowed to adhere to COL4 for 15 h and pulsed with methyl-[3H]thymidine for the last 3 h. Data are expressed as mean ± S.E. of triplicate wells. A representative experiment of three is shown.

 
The abnormal proliferative response following PC-3 cell adhesion onto BM was linked to a sustained BRCA2 protein depletion and could be completely averted by wild-type BRCA2 cDNA transfection. This pathway seems to switch cell proliferation on and off depending on a critical amount of BRCA2 protein, as suggested by the unresponsiveness to mild reductions in BRCA2 protein after PC-3 cell adhesion to FN. Conversely, normal cell adhesion to FN resulted in an increase in BRCA2 expression with no discernible effect upon DNA synthesis. Our findings add to an increasing body of evidence suggesting that BRCA2 also functions as modulator of cell proliferation and tumor growth. A previous report showed that recombinant BRCA2 overexpression in the BRCA2-deficient pancreatic adenocarcinoma cell line Capan-1 resulted in substantial decrease in tumorigenicity when inoculated into nude mice (31). More recently, Miyamoto et al. (32) observed that Capan-1 cells exhibited an increased proliferation after adhesion to COL4. The mechanisms involved are not yet well understood. Normally, part of the cellular response to DNA damage involves the activation of an ATM/p53/Mdm2 feedback loop that regulates cell cycle progression and/or apoptosis in response to relative amounts of a DNA repair complex containing BRCA2 (33-35). However, involvement of this mechanism seems unlikely because our experiments demonstrate no relationship between beta1 integrin signaling and Mdm2 levels and suggest an alternative pathway involving Skp2 (see below). Furthermore, PC-3 (and Capan-1) cells exhibit inactivating mutations in the p53 gene (36, 37).


Figure 8
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  		FIGURE 8.
Hypothesized role of BRCA2 in BM-dependent regulation of cancer cell proliferation. BM-dependent signals via beta1 integrins activate PI 3-kinase, increasing Skp2 protein levels. Skp2 interacts with BRCA2, promoting its ubiquitination (Ub-BRCA2), hence targeting it for proteasomal degradation. Ultimately, BRCA2 depletion results in increased cell proliferation at the BM interfaces.

 
Depletion of BRCA2 protein upon cancer cell adhesion to BM proteins and osseous COL1 (24) occurs through protein degradation in the proteasome. However, the precise mechanisms involved had not been elucidated before. In the present study, we have provided evidence that this event is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex (28). Indeed, down-regulation of Skp2 by siRNA was sufficient to rescue BRCA2 levels inhibiting the burst in cell proliferation upon cancer cell adhesion to BM. Evidence that this mechanism may be relevant in prostate cancer has also been provided in immunohistochemical studies performed on 622 radical prostatectomy specimens that demonstrate that Skp2 levels and cell-labeling frequency increase dramatically in both premalignant prostatic intraepithelial lesions and prostate carcinoma (19). Changes in Skp2 levels are dependent upon beta1 integrin-mediated signaling for Skp2 up-regulation, and subsequent BRCA2 degradation could be prevented by a beta1 integrin-blocking antibody. Evidence demonstrating that cell adhesion to the ECM results in Skp2 mRNA and protein up-regulation had been provided previously (38). We confirm those results and demonstrate for the first time that beta1 integrin-mediated signaling is necessary for ECM-dependent changes in Skp2 expression.

The up-regulation of Skp2 that follows beta1 integrin adhesion is signaled through PI 3-kinase/AKT and could be abrogated by transfecting a dominant negative form of PI 3-kinase. These observations are in agreement with recent reports implicating PI 3-kinase in Skp2-dependent degradation of p27kip1 and FOXO1 (17, 18, 39, 40). The involvement of PI 3-kinase/AKT in BRCA2 protein depletion was not surprising, as activation of this pathway has been consistently invoked in studies on cancer cell proliferation and survival (41-44). Recently, compelling confirmatory evidence for increased PI 3-kinase/AKT phosphorylation in prostate intraepithelial neoplasia and at the invasive edge of prostate carcinoma has been obtained by reverse-phase protein microarrays combined with laser microdissection (45).

Overall, our studies have demonstrated that Skp2 directly interacts with and promotes the degradation of BRCA2. As depicted in Fig. 8, this process requires PI 3-kinase/AKT activation initiated by beta1 integrin-mediated signaling following cancer cell interaction with BM proteins, a crucial physiopathological phenomenon at the beginning of the metastatic cascade to local and distant organs. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.


   FOOTNOTES
 
* This work was supported by the Ministero dell' Istruzione, Università e Ricerca-Contributi Straordinari di Ricerca/Aree Obiettivo 1 Grant (to E. M.). 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. Back

1 To whom correspondence should be addressed: Inst. of Biomembranes and Bioenergetics, National Research Council (C.N.R.), Via Amendola 165/A, 70126 Bari, Italy. Tel.: 39-080-544-2412; Fax: 39-080-544-3317; E-mail: l.moro{at}ibbe.cnr.it.

2 The abbreviations used are: BM, basement membranes; ECM, extracellular matrix; COL4, collagen type IV; LN, laminin; FN, fibronectin; PLN, perlecan; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI 3-kinase, phosphatidylinositol 3-kinase; Me2SO, dimethyl sulfoxide; E3, ubiquitin-protein isopeptide ligase; siRNA, small interference RNA. Back

3 L. Moro, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. C. Hung for the BRCA2 cDNA construct and Dr. R. Freeman for the PI 3-kinase construct.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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T. Lwin, L. A. Hazlehurst, S. Dessureault, R. Lai, W. Bai, E. Sotomayor, L. C. Moscinski, W. S. Dalton, and J. Tao
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