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[Cancer Research 59, 3230-3238, July 1, 1999]
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[Cancer Research 59, 3230-3238, July 1, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Protein Kinase C {delta} Involvement in Mammary Tumor Cell Metastasis1

Susan C. Kiley, Kimberly J. Clark, Michelle Goodnough, Danny R. Welch and Susan Jaken2

Adirondack Biomedical Research Institute, Lake Placid, New York 12946 [S. C. K., K. J. C., M. G., S. J.], and Jake Gittlen Cancer Research Institute (C7810), Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [D. R. W., S. J.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis requires cytoskeletal remodeling for migration, adhesion, and extravasation of metastatic cells. Although protein kinase C (PKC) is involved in tumor promotion/progression and cytoskeletal remodeling, its role in metastasis has not been defined. PKC{delta} levels are increased in highly metastatic 13762NF mammary tumor cells (MTLn3) compared with less metastatic, parental cell lines. To determine whether the increase in endogenous PKC{delta} is functionally related to their increased metastatic potential, we prepared MTLn3 cells that express the inhibitory regulatory domain fragment of PKC{delta} (RD{delta}) under the control of a tetracycline-inducible promoter. RD{delta} expression attenuated endogenous PKC activity, as demonstrated by decreased phosphorylation of the PKC substrate adducin in migrating cells. Thus, in MT cells, RD{delta} appears to primarily influence cytoskeleton-dependent processes rather than cell cycle progression. To determine whether RD{delta} expression influenced metastatic potential in vivo, MTLn3/RD{delta} cells were either grown in the mammary fat pad or injected into the tail vein of syngeneic rats, and effects of doxycycline-induced RD{delta} expression on pulmonary metastases were studied. Consistent with the in vitro data, induction of RD{delta} significantly reduced the number of lung metastases without affecting growth of the primary tumor. These results suggest that interfering with endogenous PKC{delta} activity by expressing the inhibitory RD{delta} fragment inhibits cytoskeleton-regulated processes important for MTLn3 cell metastasis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis is a multistep process in which tumor cells lose their contacts with neighboring cells, migrate from the primary tumor into the circulation, attach to capillary walls, escape from the vasculature, and invade and colonize other sites to establish secondary tumors (1) . To a large extent, the cellular processes associated with metastasis depend on cytoskeletal remodeling. In fact, several cytoskeletal structural and regulatory proteins, such as E-cadherin, {alpha}-catenin, ß-catenin, rac, and rho, have been implicated in metastatic progression (reviewed in (Refs. 2, 3, 4 ). Identifying molecular mechanisms that regulate the cytoskeleton-dependent processes associated with metastatic progression is key to developing new therapies to treat and prevent metastatic disease.

PKC3 is a family of phospholipid-dependent ser/thr kinases, the activity of which was linked to tumor cell promotion and progression because they are the major cellular receptors for tumor promoting phorbol esters (reviewed in Ref. 5 ). Phorbol esters cause rapid changes in cell morphology, cell-cell interactions, and cell migration, indicating that PKC activation regulates cytoskeletal reorganization (6) . PKCs also localize to discrete cytoskeletal structures, indicating that they are positioned to interact with and phosphorylate cytoskeleton-associated proteins (7, 8, 9, 10, 12) . The notion that PKC regulates cytoskeletal functions is further supported by the observation that many PKC substrates in intact cells are cytoskeleton-associated proteins (13, 14, 15, 16, 17, 18, 19) . By modifying the phosphorylation state and consequently the functions of these cytoskeleton-associated proteins, PKC activation can rapidly influence the organization of cytoskeletal structures.

Most cells express several conventional and novel PKC isozymes, all of which can be activated by phorbol esters. Although PKCs are clearly implicated in tumor promotion/progression and cytoskeletal remodeling, the specific roles, if any, of the individual isozymes have yet to be determined. One approach to studying the roles of individual PKCs in tumor progression is to identify changes in PKC isozyme expression that correlate with progressive stages of transformation. Functional relevance can then be assessed by increasing or decreasing individual PKC activities by overexpressing wild-type or dominant-negative constructs, respectively. In previous studies, we investigated the potential involvement of individual PKCs in mammary tumor progression using related cell lines with different metastatic potentials that were originally derived from 13762NF rat mammary adenocarcinomas (20 , 21) . Low and moderately metastatic cell lines were derived from tumors growing in the mammary fat pad, whereas the more rapidly growing and highly metastatic MTLn3 cell line was derived from lung metastases. Comparison of the PKC isozyme levels in three related rat MT cell lines demonstrated that PKC{delta} levels increased 3-fold in the highly metastatic MTLn3 cells compared with less metastatic cell lines (4) . To determine whether this increased PKC activity contributed to metastatic potential, we overexpressed PKC{delta} in the less metastatic cells and demonstrated that increased PKC{delta} does not affect anchorage-dependent cell growth but significantly enhances anchorage-independent growth. These studies indicated that PKC{delta} does not directly regulate cell cycle progression but may be involved in attachment-generated growth signals. In complementary studies, we found that expression of the NH2-terminal regulatory domain of PKC{delta} (RD{delta}) selectively interfered with PKC{delta} localization and activity at cell peripheries. These studies in less metastatic cells established the efficacy of RD{delta} as a PKC{delta} inhibitor and suggested a functional link between PKC{delta} activity and metastatic progression.

In the present investigation, we tested the hypothesis that excessive or inappropriate activation of PKC{delta} is functionally significant in metastasis of mammary carcinoma cell lines in vivo. To do this, we constructed cell lines from the highly metastatic MTLn3 cells with inducible expression of the inhibitory regulatory domain fragment of PKC{delta} (RD{delta}). In culture, induced expression of RD{delta} inhibited anchorage-independent growth, cell attachment, motility, and invasion but had no effect on anchorage-dependent growth of MTLn3 cells. In animals, inducing RD{delta} significantly reduced lung metastases but had no effect on growth of MTLn3 primary tumors in the mammary fat pad. Taken together, our results demonstrate that RD{delta} inhibits metastases by interfering with cytoskeleton-dependent processes that are also relevant for attachment, migration, and anchorage-independent growth of cultured cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
{alpha}-MEM, FBS, select agar, collagen type IV, and G418 sulfate were purchased from Life Technologies, Inc. (Grand Island, NY). TransIT-100 was procured from Pan Vera Corp. (Madison, WI). Mouse anti-HA monoclonal (clone 12CA5) was from Boehringer-Mannheim (Indianapolis, IN). Rabbit anti-pSer660 adducin antiserum was raised to a phosphopeptide containing the PKC phosphorylation site in {alpha}-, ß-, and {gamma}-adducins. The antiserum was affinity purified and shown to be phosphorylation-state selective (5) . Horseradish peroxidase-conjugated secondary antibodies and ECL detection reagents were from Amersham Life Science, Inc. (Arlington Heights, IL). Affinity-purified FITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and TRITC-conjugated goat anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Female Fischer 344 rats (6–7 weeks of age) were from Harlan Sprague Dawley (Indianapolis, IN). Chemicals were from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise.

Cell Culture.
MTLn3 (passages 45–47) rat 13762NF mammary adenocarcinoma cells were grown in antibiotic-free {alpha}-MEM supplemented with 5% FBS at 37°C in a humidified CO2 atmosphere, as described previously (4) .

Construction of Expression Vectors and Cell Lines.
The regulatory domain fragment of PKC{delta} (RD{delta}), including a triple repeat of the HA epitope tag sequence (H3) at the 5' end, was ligated to the BgIII/EcoRV sites of pUHD10-3 (a gift from Dr. Hermann Bujard, Zentrum für Molekulare Biologie, Heidelberg, Germany) to provide inducible expression from the minimal cytomegalovirus promoter-tetracycline operator (22) . MTLn3 (passage 45) cells were cotransfected with pUHD10-3 or pUHD10-3/H3RD{delta} and pUHD172-1neo, which encodes the reverse tetracycline transactivator and neomycin resistance, at a ratio of 10:1 using TransIT-100. G418-resistant colonies were selected, and Dox-induced HA-RD{delta} was monitored on immunoblots stained with HA antibody. Dox-inducible clones were selected and subcloned twice by limiting dilution to obtain homogeneous populations. Cells transfected with the empty vector pUHD10-3 were used to control for potential nonspecific effects of Dox treatment. Stable transfectants were maintained in {alpha}-MEM medium containing 5% FBS and 175 µg/ml G418 (complete medium).

Sample Preparation and Immunoblot Analysis.
For screening RD{delta} clones, cells in duplicate wells (± Dox treatment) of a 24-well tray were scraped into 150 µl of boiling Laemmli Buffer (23) . Cell lysates from 100-mm dish cultures were prepared as described (24) . Tissue homogenates were prepared on ice with a handheld tissue homogenizer, using 150–200 mg of frozen tissue/ml homogenization buffer [20 mM Tris-HCl (pH 7.4), containing 0.25 M sucrose, 5 mM EDTA, 10 µg/ml leupeptin, 100 µg/ml aprotinin, 50 mM sodium fluoride, and 1 mM sodium vanadate]. Samples were normalized by protein concentration and solubilized in boiling Laemmli buffer. Cell/tissue proteins were separated on 10% acrylamide gels by SDS-PAGE, electrophoretically transferred to nitrocellulose, and stained with antibodies, as described previously (24) . The HA monoclonal (12CA5) antibody was used at 1 µg/ml in TBA [20 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl and 1% w/v BSA]. Immunoreactive bands were visualized with ECL reagents according to the manufacturer’s recommendations.

Immunofluorescence in Whole Cells.
To monitor RD{delta} expression in cells, MTLn3/RD{delta} cells were grown to 40–50% confluence and then incubated with ±1 µg/ml Dox for 20–24 h. Cells were fixed in 3.7% formaldehyde for 5 min at room temperature and permeabilized in -20°C methanol for 3 min. Immunostaining procedures were as described previously (24) . The HA antibody was used at 3.3 µg/ml in PBS containing 1% w/v BSA. After washing, coverslips were incubated with FITC-conjugated goat anti-mouse (1:300) antibodies for 1 h. Slides were observed on a Nikon Optiphot fluorescence microscope equipped with a x100 objective lens, and photographs were taken with Kodak TMAX 400 black and white film.

Anchorage-dependent Growth.
For growth rate studies, 104 cells in complete medium were seeded into wells of 6-well trays on day -1 and allowed to attach overnight. The next day (day 0), medium was replaced with complete medium without or with 1 µg/ml Dox to initiate RD{delta} expression. Triplicate wells were trypsinized and counted daily.

Clonogenic Growth in Soft Agar.
Uninduced cells (104) in complete medium ± Dox containing 0.33% top agar were plated into triplicate 35-mm wells containing solidified 0.66% agar. Where indicated, Dox (1 µg/ml) was included throughout the 2-week growth assay. At the end of 2 weeks, colonies were stained and counted as described.4

Cell Attachment Assay.
A microtiter plate assay described by Hansen et al. (25) was modified and used to monitor the effect of RD{delta} transgene expression on MTLn3 cell attachment. Cells (± Dox pretreatment) were detached with trypsin and resuspended to a final concentration of 5 x 105 cells/ml in complete medium. These cells were replated into microtiter plate wells (5 x 104 cells/well) coated with collagen type IV (30 µl of 10 µg of collagen/ml PBS per well). Where indicated, Dox (1 µg/ml) was added back to the Dox-pretreated cells, and cells were incubated at 37°C in a CO2 incubator for 30 min to 8 h. At each time point, unattached cells were removed, and the vital dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide was added (1 mg/ml). Cells were incubated for an additional 2 h at 37°C for complete color development, and attached cell number was estimated according to absorbance at 570 nm as described previously.4

Zymography.
Vector control and RD{delta} cell cultures at 80% confluence were rinsed once with PBS and then incubated in the absence or presence of 1 µg/ml Dox in 5 ml of serum-free {alpha}-MEM containing 10 mM HEPES (pH 7.4) and 5 µg/ml insulin for 16 h. Cell-free conditioned medium (3 ml) was concentrated to 50 µl in Centricon-30 filtration units (Amicon, Inc., Beverly, MA). Aliquots (25 µl) were mixed with nonreducing Laemmli buffer and electrophoresed through collagen-embedded gels as described (26) . After electrophoresis, the collagen/acrylamide gel was washed, incubated at 37°C to activate the proteases, and stained according to described methods to visualize regions of digested collagen (26) .

Invasion Assay.
Dox effects on invasive capacity were assayed by plating cells (± Dox pretreatment) into the upper well of Costar Transwell units in serum-free medium and monitoring cell migration through ECM-coated filters toward FBS-supplemented medium ± Dox. The upper side of the Transwell membrane (pore size, 8.0 µm) was coated with collagen type IV (0.25 µg of collagen/ml PBS per well). Cells were grown in the absence or presence of 1 µg/ml Dox for 24 h, harvested with trypsin, and resuspended in serum-free {alpha}-MEM containing 10 mm HEPES (pH 7.4) and 0.1% w/v BSA. The number of cells/cm2 seeded to Transwells was doubled (relative to the number of cells used in attachment assays) to compensate for the decreased efficiency of RD{delta} cell attachment (± Dox). Aliquots (105 cells) were plated into the upper well of the collagen-coated Transwell inserts placed into wells containing media ± FBS and ± Dox. Cells were incubated for 40 h at 37°C, medium was aspirated, and cells on the upper side of the membrane were removed with a cotton swab. The lower well and underside of Transwell membrane were washed twice with PBS before cells were fixed with 5% formaldehyde in PBS for 10 min. Cells were stained with crystal violet and counted at x200. Four fields were counted for each of the triplicate membranes.

In Vivo Metastasis Assays.
To determine the effect of RD{delta} on MTLn3 cell metastasis, parental and variant MTLn3 cells were injected into the mammary fat pad or into the tail vein (20 , 26) . MTLn3 (passage 45) or MTLn3/RD{delta} clone 51 (passage 5) cells were cultured in the absence or presence of 1 µg/ml Dox for 24 h. Cells were trypsinized, counted, washed twice, and resuspended in ice-cold sterile PBS. For mammary fat pad injections, animals were anesthetized lightly with Ketaset Rompun and injected s.c. with 106 viable cells in 0.5 ml of PBS. Where indicated, Dox (200 µg/ml in 2.5% sucrose) was administered in the drinking water. Tumors were palpitated every 3–4 days after the first week and measured with calipers (mean tumor diameter, square root of tumor length x width, in mm) to quantitate tumor growth. Animals were killed with Euthasol on day 25 after tumor cell injection. Tumors growing at the site of injection were measured and then removed and rinsed in ice-cold PBS. Pieces of tumor tissue were rapidly frozen in liquid nitrogen and subsequently used to prepare tissue homogenates for immunoblot analysis. Lungs were removed, rinsed in ice-cold PBS, and fixed in Bouin’s solution for 24 h. Metastatic lesions were counted using a dissecting microscope.

For tail vein injections, 105 cells in 0.2 ml PBS were injected into the lateral tail vein. Where indicated, Dox was administered in the water supply as described above. Animals were sacrificed with metofane overdose on day 9 after injection. Lungs were removed, rinsed in cold PBS, and fixed in Bouin’s solution to quantitate surface lesions.

Cell Migration and Adducin Phosphorylation.
To monitor the effect of RD{delta} expression on PKC-mediated phosphorylation of adducin during cell migration, cells were grown to 80% confluence in 24-well trays and on coverslips. Where indicated, 1 µg/ml Dox was added to cultures for 24 h before scraping an artificial wound into the cell monolayer. Monolayers were washed once with PBS to remove dislodged cells, and medium was replenished. Dox was added back to cultures that had been pretreated with Dox for 24 h, and aphidicolin (10 µg/ml) was added to half the cultures to inhibit cell growth. At 6, 12, 18, and 24 h after wounding the monolayer, cells in 24-well trays were rinsed with PBS, fixed in 5% PBS-buffered formaldehyde for 10 min, and stained with 0.1% w/v Crystal Violet for 5 min. There was no evidence of aphidicolin toxicity at the 24-h time point. The 24-well plates were individually photographed and printed on 8 x 10-inch paper for enlargement so that the wound diameter could be measured with a ruler.

Coverslips were fixed as described previously at 6, 12, and 24 h after wounding the cell monolayer and incubated with affinity-purified rabbit pSer660-adducin antibody (20 µg/ml) and mouse anti-HA antibody (0.33 µg/ml) for double-label immunostaining. After washing, coverslips were incubated with FITC-conjugated goat anti-rabbit IgG (1:300) and TRITC-conjugated goat anti-mouse IgG (1:5000) antibodies. Coverslips were observed on a Nikon fluorescence microscope equipped with a x100 objective lens and appropriate filters for fluorescein and rhodamine.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RD{delta}d Expression in MTLn3 Clonal Cell Lines.
In previous studies, we found that PKC{delta} message and protein levels were increased in highly metastatic MTLn3 cells compared with related, less metastatic MT cell lines.4 To determine whether the increase in PKC{delta} plays a functional role in promoting MT cell growth and metastasis, we generated clones of MTLn3 cells expressing the inhibitory (dominant-negative) regulatory domain fragment of PKC{delta} (RD{delta}). The construct includes the HA epitope-tag sequence and was expressed from a tetracycline-inducible promoter system (22) . MTLn3/RD{delta} clones were selected for G418 resistance and cloned by limiting dilution. The clones used in these studies express minimal levels of HA-RD{delta} in the absence of Dox and >=10-fold higher levels in the presence of Dox (Fig. 1)Citation . Induction of RD{delta} was dose dependent, and Dox was not toxic at the concentration used (data not shown; however, note that Dox did not inhibit soft agar growth, invasion, or migration of vector control cells; Tables 1Citation , 2Citation , and 5Citation ). Inducible homogeneous RD{delta} expression was retained by all clones for at least eight passages (through passage 15 for clone 51), and all experiments were performed with passage 4 to 7 cells unless indicated otherwise. "Vector" refers to MTLn3 cells cotransfected with empty expression plasmid and the neomycin-resistance plasmid, which were then selected for G418 resistance. Vector cells were not significantly different from wt MTLn3 cells with respect to cell attachment, collagenase activity, or invasion, indicating that transfection and expression of the tetracycline regulator had no significant effects on the behavior of cultured MTLn3 cells (Table 2Citation and data not shown).



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  		Fig. 1. Expression of the epitope-tagged RD{delta} construct. Left, cell lysates were prepared from duplicate cultures of G418-resistant MTLn3 cell lines grown in the absence (-) or presence (+) of 1 µg/ml Dox for 24 h. Cell extracts (50 µg of protein) from vector control and RD{delta} clone 21 were separated by SDS-PAGE and blotted to nitrocellulose. Immunoreactive bands were identified by probing blots with antibody directed toward the HA epitope tag sequence (arrowhead). Left, molecular weight standards (in thousands). Films from blots were scanned on a densitometer and quantitated according to absorbance. Right, MTLn3/RD{delta} 21 cells grown on coverslips were treated ± 1 µg/ml Dox for 24 h. Cells were fixed and stained with anti-HA monoclonal antibody and FITC-conjugated goat anti-mouse IgG, as described in "Materials and Methods." Photographs were taken with a x100 objective lens. Identical results were obtained in at least three experiments.

 

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  		Table 1 Effect of RD{delta} on MTLn3 cell clonogenic growth in soft agar

 

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  		Table 2 Effect of RD{delta} on invasive properties of MTLn3 cells in vitro: Invasion into chamber containing medium supplemented with 5% FBS

 

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  		Table 5 Effect of RD{delta} expression on migration into an artificial wound

 
Effects of RD{delta}d Expression on MTLn3 Cell Growth and Attachment.
To determine whether the inhibitory RD{delta} construct directly influences cell growth, we compared the growth kinetics of vector control and RD{delta}-expressing MTLn3 cells (Fig. 2)Citation . Growth rates and doubling times were similar in the presence or absence of Dox, indicating that neither Dox nor RD{delta} interfere with cell growth. The number of RD{delta} cells (-Dox) on day 1 was consistently lower than the number of cells seeded, and Dox potentiated this decrease. This did not occur with vector control cells, which suggests that the RD{delta} cell lines do not attach to plastic as efficiently as vector control cells. Induced RD{delta} expression caused a small but significant decrease in saturation density from 9.5 x 105 to 7.1 x 105 cells/cm2. These results demonstrate that RD{delta} does not directly influence growth rate but may affect other cell functions, such as substratum attachment.



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  		Fig. 2. Effect of RD{delta} expression on MTLn3 cell growth. Cells (without Dox pretreatment) were seeded at 104 cells/well (dashed horizontal line) in six-well trays on day -1. After attaching overnight, medium was replaced with medium ± Dox (day 0), and cells were grown in the absence (•) or presence ({circ}) of Dox for the indicated times. Growth rates and saturation densities were compared in cells transfected with the empty vector (top) and RD{delta} (clone 51, bottom). Cell numbers were determined in triplicate, and the curves shown are representative for vector control and all three RD{delta} clones in two independent experiments.

 
To directly test the effects of RD{delta} on attachment, we monitored wt- and RD{delta}-MTLn3 cell attachment to four different ECM proteins. After a 1-h incubation, >60% of the wt MTLn3 cells attached collagen type IV-coated dishes, whereas only 30–40% attached to laminin- or Matrigel-coated dishes, and <10% attached to fibronectin-coated dishes. Dox slightly increased attachment of wt and vector control MTLn3 cells, but this effect was relatively small (<10%). For vector control cells, 95% of the cells were attached 4 h after plating on collagen, and Dox pretreatment did not influence attachment (Fig. 3Citation , top). In contrast, even without Dox pretreatment, <50% of the RD{delta}21 and <20% of the RD{delta}51 cells were attached 8 h after plating (Fig. 3)Citation . Treatment with Dox further reduced cell attachment to 25 and 7.5% for RD{delta}21 and RD{delta}51 clones, respectively. The difference between the vector control, RD{delta}21 cells, and RD{delta}51 cells in the absence of Dox treatment may be due to a low level leakiness of the expression system. Dox-induced inhibition of attachment in RD{delta} but not vector control cultures provides strong evidence that RD{delta} interferes with molecular events required for cell substratum attachment in MT cells.



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  		Fig. 3. Effect of RD{delta} on cell attachment. MTLn3 cell cultures were preincubated in the absence (•) or presence ({circ}) of Dox for 24 h to induce expression of the inhibitory RD{delta} fragment. Cells were trypsinized, and 5 x 104 cells were plated into microtiter trays precoated with collagen type IV. Attached cells were quantitated as described in "Materials and Methods" and expressed as a percentage of total cells/well. Identical results were obtained in two additional experiments.

 
Effect of Ectopic RD{delta} Expression on Clonogenic Growth in Soft Agar.
Increased growth of cultured cells in soft agar often correlates with increased metastatic potential in vivo. To determine whether dominant-negative RD{delta} could inhibit growth in soft agar, we assayed anchorage-independent growth of vector control and RD{delta} cells in the absence or presence of Dox. Dox did not affect the number of vector control colonies formed in semisolid medium (Table 1)Citation . In contrast, Dox-induced RD{delta} expression reduced the number of colonies to 24–37% of the corresponding control cultures (Table 1)Citation . These results support the hypothesis that RD{delta} expression inhibits processes that correlate with metastatic progression in vivo.

Effect of RD{delta}d Expression on Collagenolytic Activity and Cell Invasion.
Metastatic cells must degrade ECM barriers to enter and exit from the vasculature and colonize secondary tissues. Invasion is in part mediated through secretion of metalloproteinases that break down ECM. To determine whether RD{delta} expression influences metalloproteinase levels, we monitored secreted collagenase activity by zymography. The type IV collagenolytic activities detected in vector control and three RD{delta} cultures were identical with or without Dox treatment (Fig. 4)Citation . These results indicate that RD{delta} expression has no measurable effect on metalloproteinase secretion or activity.



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  		Fig. 4. Effect of RD{delta} on secreted type IV collagenolytic activity. Vector control and three different RD{delta} clones were cultured in the absence (-) or presence (+) of Dox for 16 h. Samples were prepared from the concentrated conditioned medium collected from each culture and electrophoresed into a collagen type IV-embedded acrylamide gel as described in "Materials and Methods." The gel was incubated at 37°C for 16 h to activate immobilized proteases and stained with Coomassie blue. Type IV collagenolytic activity is indicated by transparent bands in the collagen-embedded gel (arrowhead on the right). Left, molecular weight standards (in thousands). These results were verified in a second experiment.

 
Cell motility is another cytoskeleton-dependent process required for metastasis. We used an in vitro assay for cell migration through collagen-coated membranes to evaluate the effect of RD{delta} expression on MTLn3 cell motility. In this assay, invasion involves attachment to and migration through ECM-coated filters toward FBS-supplemented medium in the lower chamber. Dox treatment profoundly inhibited invasion of RD{delta}21 and RD{delta}51 clones, reducing the number of invading cells from 200 to 300 to <2 cells/field (Table 2)Citation . Because RD{delta} expression did not reduce metalloproteinase activity, these results indicate that RD{delta} interferes with other cellular processes relevant to invasion, such as cell motility.

Effect of RD{delta}d Expression on MT Growth and the Development of Metastatic Lesions.
The studies with cultured cells indicate that although RD{delta} expression does not influence cell growth rates, it does interfere with several correlates of tumor progression and metastasis: anchorage-independent growth, cell attachment, motility, and invasion. However, because metastasis is a complex series of processes, metastatic potential cannot be fully evaluated in cell culture studies; animal studies are needed for complete evaluation of metastatic potential. To directly determine whether RD{delta} expression would alter MTLn3 cell tumor growth or metastases, wt and RD{delta}51 cells were transplanted into the mammary fat pad of female syngeneic rats. Where indicated, RD{delta} was induced by treating the cells in culture with Dox for 24 h before harvesting and/or by administering Dox to the rats through their drinking water (in culture/in vivo, Table 3Citation ). 2Animals were sacrificed on day 25, at which time primary tumors and lungs were harvested for further analysis. Macroscopic lung metastases are usually not apparent before day 25, although micrometastases may be observed earlier. The 25-day lag time between transplanting cells into the fat pad and identifying surface lung lesions reflects the time required for MTLn3 cells to migrate from the primary tumor site in the fat pad to the lungs and expand into a visible colony.


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  		Table 3 Effect of RD{delta} expression on MTLn3 tumor growth and lung metastasis

 
In experiment 1, we compared the effects of Dox on metastasis derived from wt and RD{delta}51 cells (Table 3A)Citation . Dox did not influence either the size of tumors or the number of metastases derived from wt cells, indicating that Dox treatment alone does not induce responses that interfere with MTLn3 tumor growth or metastasis (Table 3A)Citation . In the absence of Dox, tumor size and number of metastases for RD{delta}51 cells were not different from the wt cells. These results establish that neither Dox nor transfection per se significantly influence the metastatic potential of these cells.

In contrast to the wt cells, Dox treatment significantly decreased metastasis of the RD{delta}51 tumors (Table 3A)Citation . The number of lung lesions was reduced from a median of 140 to 3 in Dox-treated animals. Dox-induced inhibition correlated with Dox-induced HA-RD{delta} expression in the RD{delta}51 tumors (Fig. 5A)Citation . On the other hand, Dox did not influence RD{delta}51 tumor sizes, a result that is consistent with the observation that induced RD{delta} does not inhibit anchorage-dependent growth of cultured RD{delta}51 cells (Fig. 2)Citation . Thus, the main effect of RD{delta} is inhibition of metastasis rather than primary tumor growth.



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  		Fig. 5. Effect of Dox treatment duration on RD{delta} expression in mammary tumors and the development of pulmonary metastases. Where indicated by (+), Dox was administered to cells in culture, 24 h before injection into mammary fat pad, or was added to the drinking water from 3 days prior to injections (day -3) to the end of the experiment (day 25); actual initiation and duration of in vivo treatments are shown in parentheses for each group. On day 25, animals were sacrificed, and tissues were harvested. In A, tumor tissue extracts (50 µg of protein) were separated on SDS-PAGE and blotted to nitrocellulose; HA-tagged RD{delta} was detected by probing blots with HA-specific antibody (arrowhead). Left, molecular weight standards. In B, lungs harvested on day 25 were fixed in Bouin’s solution, and surface lesions were counted using a dissecting microscope. The lungs shown are representative of the nine collected for the -/- and +/+ (day -3 to 25) groups.

 
In a second experiment, the mechanism of RD{delta} inhibition was investigated further by varying the Dox treatment intervals (Table 3B)Citation . We compared the effects of continual to partial (-3 to 14 or -3 to 7 days) Dox treatment on tumor size, metastasis, and RD{delta} expression. As in experiment 1, continual Dox treatment significantly inhibited metastasis without inhibiting growth of the mammary tumor (Table 3BCitation and Fig. 5BCitation ). RD{delta} levels in day-25 tumors from the animals receiving the full course Dox treatment were high. In contrast, levels in day-25 tumors from animals receiving Dox treatment only up to day 7 or day 14 were reduced to the levels in tumors from animals that did not receive Dox, demonstrating that Dox-dependent RD{delta} induction is reversible in vivo (Fig. 5A)Citation . Despite the reduced RD{delta} levels, pulmonary metastasis were significantly decreased in these animals. This is likely to be due to the significant lag time between establishment and growth of a micrometastatic lesion to a visible colony. It is likely that the effect of Dox reversal would become more apparent at later times. However, the tumor burden at day 25 does not permit extending the experiments beyond this time.

In each of the protocols described above, cultured cells were pretreated with Dox before transplantation into the fat pad. Because Dox treatment inhibits cell attachment, inhibition of metastasis could be due to decreased attachment of injected cells in the mammary fat pad. However, because pretreatment with Dox does not significantly inhibit tumor growth, this does not seem likely. Nonetheless, to directly determine whether Dox pretreatment was necessary for the reduction in metastatic potential, RD{delta}51 cells that were not pretreated with Dox were transplanted into the fat pad of animals that were not primed with Dox. Dox treatment was begun on day 7, at which time a palpable tumor had formed (-/+, 7–25 days, Table 3BCitation ). Even under these conditions of delayed Dox treatment, RD{delta} expression significantly attenuated the development of metastatic lesions on the lungs (Table 3B)Citation . These results demonstrate that RD{delta} expression interferes with metastatic progression rather than establishment of the primary tumor. On the basis of the effects of RD{delta} expression on properties of cultured MTLn3 cells, it is likely that the reduced metastatic potential of Dox-treated RD{delta}51 cells is related to their decreased anchorage independence, invasion, motility, and/or substratum attachment.

In a second series of experiments, MT cells were directly injected into the tail vein, and lung metastases were evaluated 9 days later. In this assay, metastasis depends on the ability of the injected cells to survive in the circulation, escape from pulmonary capillaries, attach to lung tissue, and grow in that environment. All animals injected with control or Dox-treated wt cells produced large numbers of lung metastases (Table 4)Citation . Animals injected with uninduced RD{delta}51 cells also produced a large number of lung metastases, again indicating that uninduced RD{delta} cells are not significantly different from wt cells in vivo. In contrast to the wt cells, Dox-pretreated RD{delta}51 cells injected into syngeneic animals (with or without Dox treatment) formed relatively few metastatic lung lesions (+/- and +/+, Table 4Citation ). These experiments indicate that expression of RD{delta} during the first 24–48 h after injection critically influences survival in the circulation, attachment, invasion, and/or colonization of lung tissue. Thus, both the mammary fat pad and tail vein injection protocols indicate that the principal effect of RD{delta} expression in vivo is the suppression of lung colonization by MTLn3 cells.


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  		Table 4 Effect of Dox treatment and RD{delta} on the development of lung metastases derived from MTLn3 cells injected into the tail vein

 
Effect of RD{delta} on Cell Migration and Adducin Phosphorylation in Situ.
The predicted mechanism of attenuation of metastases by RD{delta} in vivo is through inhibition of PKC substrate phosphorylation. In vitro, RD{delta} inhibits PKC catalytic activity and PKC binding to its substrates (27) ;5 however, in vivo inhibition of substrate phosphorylation has not yet been demonstrated. We therefore monitored the effects of RD{delta} expression on the phosphorylation state of a known PKC substrate, adducin. In previous studies, we demonstrated that adducin phosphorylation was increased in cells induced to migrate into an artificial wound.5 Wounding stimulates both cell growth and migration; therefore, aphidicolin was added to cultures to arrest cells in S-phase and minimize the effects of growth on our measurements of wound closure and adducin phosphorylation. Migration was assessed by measuring the decreased width of the scraped area in photomicrographs. Wound widths of vector control (± Dox) and RD{delta}51 cells without Dox treatment were similar, indicating that Dox treatment did not interfere with cell migration (Table 5)Citation . In contrast, Dox significantly increased wound widths of the RD{delta}51 cells, indicating that Dox-induced RD{delta} interfered with migration (Table 5)Citation . The contribution of cell growth to wound closure is apparent in the decreased wound width of cultures grown in the absence of aphidicolin compared with cultures grown in the presence of aphidicolin (Table 5)Citation . These results indicate that RD{delta} expression inhibits migration and that the inhibitory effect of RD{delta} is independent of cell growth.

To determine whether the RD{delta} effects on cell migration could be due in part to the inhibition of substrate phosphorylation, phosphoadducin levels were monitored with an antibody that selectively recognizes adducins phosphorylated at their PKC-specific phosphorylation site.5 pSer660-adducin has been used previously as a reporter to monitor endogenous PKC activity (16 , 17) . For these studies, 12 h after scrape wounding, cells were fixed and doubly stained with antibodies to detect phosphoadducin and HA-RD{delta} in the same cells. In the absence of Dox treatment, RD{delta} cells at the edge of the wound adopt a migratory morphology with characteristic leading lamellipodia that extended into the scraped area. Vector control cells, which looked identical, are not shown. pSer660-adducin staining is concentrated in the leading edge of the lamellipodia, although substantial cytoplasmic, perinuclear fluorescence is also observed (Fig. 6Citation , upper left). Treatment with Dox significantly decreased pSer660-adducin staining intensity in RD{delta}21 cells (Fig. 6Citation , middle). Furthermore, these cells did not display leading lamellipodia, consistent with the decrease in cell migration (Table 5)Citation . Identical results were seen with RD{delta}d51 cells and are therefore not shown. The effects of RD{delta} are emphasized in high-passage RD{delta}21 cells that have become heterogeneous with respect to inducible RD{delta} expression (Fig. 6Citation , right). Cells that no longer express RD{delta} have increased pSer660-adducin staining compared with RD{delta}-expressing cells in the same field. Treatment with Dox had no effect on pSer660-adducin levels in the vector control cells (data not shown), consistent with the results shown in Fig. 6Citation , which demonstrate that Dox selectively affects phosphorylation in the RD{delta}d-expressing cells within the heterogeneous RD{delta}d21 population. Furthermore, leading lamellipodia can be seen in the nonexpressing cells, whereas these structures are not apparent in the RD{delta}-expressing cells. Low levels of phosphoadducin in RD{delta}-positive cells were not due to reduced adducin levels, because {alpha}a- and {gamma}g-adducin levels in lysates of cells cultured in the presence or absence of Dox did not differ by >15%, as determined by densitometry of immunoblots (data not shown). The fact that RD{delta} suppresses both Ser660-adducin phosphorylation and cell migration strongly suggests a functional link between PKC{delta} activity, adducin phosphorylation, and migration.



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  		Fig. 6. Effect of RD{delta} expression on PKC-mediated adducin phosphorylation in migrating cells. MTLn3 vector control and RD{delta}21 cells that conditionally express RD{delta} were grown on coverslips to 80% confluence and were then cultured in the absence (-Dox) or presence (+Dox) of Dox for 24 h. An artificial wound was then scraped into each cell monolayer. Dox was maintained in cultures pretreated with Dox, and coverslips were incubated for 12 h. Cells were fixed and stained with anti-pSer660-adducin (+FITC-conjugated second antibodies) and anti-HA (+TRITC-conjugated second antibodies) to identify RD{delta}-positive cells in double-label immunofluorescence, as described in "Materials and Methods." Paired photographs were taken with a x100 objective lens with the open wound on the left side of each photograph. The photographs shown represent the results obtained in four independent experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mammary cancer is a disease in which events early in life, such as age of menarche and first pregnancy, dictate risk of disease decades later. Typically, early diagnosis correlates with favorable clinical outcome, indicating that tumor cells quickly progress to the metastatic phenotype. Clearly, a better understanding of processes that regulate the long latency period, promotion, and progression are needed to improve management of this disease. Progression from a nonmetastatic to a metastatic tumor cell involves a series of events that include changes in growth, gene expression, morphology, adhesion, and motility. PKC is known to regulate each of these events as well as tumor promotion in general. Thus, the purpose of this study was to test the hypothesis that the elevated PKC{delta} levels detected in highly metastatic MTLn3 cells contribute to the increased metastatic potential of those cells. One prediction of this hypothesis is that overexpressing PKC{delta} in less metastatic cells should promote their transformation. In fact, in a previous study, we demonstrated that expressing PKC{delta} in cells of moderate metastatic potential increased anchorage-independent growth (4) . A second prediction is that down-modulating PKC{delta} activity in the highly metastatic cells should inhibit their malignant behavior. In the present investigation, we expressed the inhibitory RD{delta} fragment in the highly metastatic cells and demonstrated that RD{delta} inhibited transformation in cultured cells and lung metastasis in animals. These complementary studies point to an important role for PKC{delta} as a determinant of MT cell metastatic potential.

Expressing the regulatory domain of PKC{delta} to decrease endogenous PKC{delta} activity is somewhat different from most other studies that have used catalytically inactive PKC{delta} as a dominant-negative inhibitor (28, 29, 30, 31) . RD{delta} was used because recombinant RDs are very efficacious PKC inhibitors in vitro (27) .5 As shown in this study, expressed RD{delta} was also an effective PKC inhibitor in cells. Furthermore, studies with PKC regulatory/catalytic domain chimeras and regulatory domain fragments have demonstrated that the regulatory domains play a role as isozyme-selective determinants of substrate recognition and subcellular targeting (32, 33, 34, 35, 36) . Taken together, these results provide a firm rationale for using RD expression to inhibit endogenous PKCs, although further evidence is needed to establish the extent to which inhibition is isozyme selective.

The effects of RD{delta} on attachment, migration, and invasion all indicate that PKC{delta} and possibly other PKCs regulate cytoskeletal processes involved in cell adhesion and motility. Although other studies have implicated PKC activity in attachment and migration (37, 38, 39) , our studies are the first to link PKC{delta} to these processes. We identified previously a group of cytoskeleton-associated PKC substrates called STICKs (reviewed in Ref. 13 ). Many STICKs are cytoskeleton-associated proteins, and inhibition of their phosphorylation establishes a mechanism by which RD{delta} can interfere with cytoskeleton-dependent processes, such as migration in cultured cells and metastasis in vivo. We studied adducin as an example of a STICK known to be involved in cytoskeletal remodeling. Adducins are barbed-end, actin-capping proteins, and PKC phosphorylation inhibits their actin-capping activity (40) . Thus, localized phosphorylation would be predicted to result in localized uncapping and filament elongation, processes required for lamellipodia formation and migration. We found that in the absence of RDS expression, pSer660-adducin was concentrated in leading lamellipodia of migrating MTLn3 cells and induced expression of RD{delta} reduced pSer660-adducin levels. RD{delta} effects on cytoskeletal functions can, therefore, be linked to inhibition of phosphorylation of PKC substrates such as adducin and other STICKs.

It is important to note that Dox did not significantly decrease pSer660-adducin levels in primary tumors as determined by immunoblots (Fig. 5)Citation and verified by immunohistochemical staining (data not shown). Histological sections of tumor tissue from both Dox-treated and control animals showed substantial pSer660-adducin levels, especially in cells at the tumor perimeter. Thus, it seems unlikely that decreased phosphorylation of substrates in the primary tumor accounts for the Dox-induced RD{delta} inhibition of metastasis. On the other hand, RD{delta} expression was also a potent inhibitor of metastasis of cells injected into the tail vein. To form a metastatic colony, these injected cells, as well as tumor cells that escape from the primary tumor, must survive in the circulation, extravasate, and attach to lung tissue. Because RD{delta} inhibits anchorage-independent growth/survival, attachment, and invasion in vitro, it seems likely that RD{delta} expression in vivo interferes with any or all of these processes rather than escape from the primary tumor.

Increased activity of other signaling pathways has also been linked to increased metastatic potential. In particular, increased rho, rac, and phosphatidylinositol 3-kinase activities have been linked to increased migration and invasion (41, 42, 43) . Changes in levels of specific integrins or cadherins that regulate interactions with ECM or adjacent cells, respectively, have also been linked to increased metastatic potential (2 , 3 , 44) . Our results indicate that PKC{delta} should be included among those pathways that regulate metastatic processes. Additional studies on PKC{delta} substrates and their functions will provide mechanistic insight as to how PKC{delta} regulates cytoskeleton-dependent processes associated with metastasis. Furthermore, properties of the substrates may also provide insight into how these different signaling pathways can converge on metastatic regulation. For example, recent studies have demonstrated that PKC, PKA, and rho-kinase phosphorylate adducin at different sites (45 , 46) . These distinct phosphorylation events differentially modulate adducin functions with regard to actin capping and polymerization. The net phosphorylation state of adducin at discrete subcellular sites is therefore a determinant of actin polymerization dynamics. In this way, adducin integrates signals from various pathways.

Our studies demonstrate that PKC{delta} signaling is an important determinant of metastatic potential in the 13762NF rat mammary metastasis model but do not yet address the general significance of PKC{delta} in human mammary or other cancers. Relatively little information on PKC isozyme levels in human mammary tissues is presently available. Monitoring PKC isozyme levels in human tissue samples is complicated by technical problems including the fact that PKCs can be rapidly degraded during sample collection. Furthermore, the relationship between PKC protein levels and endogenous activity is confused by the fact that active PKCs are down-regulated by proteolysis. Thus, in some cases, decreased PKC protein levels may actually correlate with increased activity (47) . An alternative to monitoring PKC isozyme levels is to use phosphorylation state-selective antibodies to monitor substrate phosphorylation levels as reporters that reflect endogenous PKC activity levels. Immunohistochemical applications of these antibodies in prospective and retrospective studies of banked human tissues should be valuable for unraveling the role of PKC in human mammary tumor progression. Nonetheless, these initial studies in rat cells clearly establish a role for PKC{delta} in regulating cytoskeletal protein phosphorylation and cytoskeleton-dependent processes relevant to in vivo metastatic potential. Thus, PKC{delta} may be considered as a potential target for designing isozyme-selective synthetic inhibitors that could be used in combination with growth-limiting therapies to control the spread of metastases.


   ACKNOWLEDGMENTS
 
We thank Chris Chapline, Lynn Fowler, Maggie Tobin, and Yi Zeng for their individual contributions to the development and characterization of the pSer660-adducin antibodies. Carol Spierto, Pamuela Murphy, Deanna Hicks, and Steven Goldberg provided expert assistance in animal management, anesthesia, and dissection. Marina LaDuke assisted in the preparation of figures for the manuscript.


   FOOTNOTES
 
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.

1 This work was supported by United States Army Medical Research and Materiel Command Grant DAMD17-94-J4312 (to S. J.), Grant DAMD17-96-1-6152 (to D. R. W.), and NIH Grant CA 71607 (to S. J.). Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Medical Alumni building, A249, University of Vermont, Burlington, VT 05405. Phone: (802) 656-9496; Fax: (802) 656-8892; E-mail: sjaken{at}zoo.uvm.edu Back

3 The abbreviations used are: PKC, protein kinase C; FBS, fetal bovine serum; TRITC, tetramethyl rhodamine isothiocyanate; Dox, doxycycline; ECM, extracellular matrix; MT, mammary tumor; HA, hemagglutinin antigen; pSer660, phosphoserine-660; STICK, substrate that interacts with C-kinase; wt, wild type. Back

4 S. C. Kiley, K. J. Clark, S. K. Duddy, D. R. Welch, and S. Jaken. Protein kinase C {delta} promotes mammary tumor cell progression toward a metastatic phenotype, submitted for publication. Back

5 C. Chapline, J. Cotton, M. Tobin, Y. Zeng, and S. Jaken, manuscript in preparation. Back

Received 12/10/98. Accepted 5/ 3/99.


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 DISCUSSION
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