Malignant conversion of non-tumorigenic murine skin keratinocytes overexpressing PACE4

Haleh Mahloogi, Daniel E. Bassi and Andres J.P. Klein-Szanto,1

Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Proprotein convertases (PCs) have been implicated in tumor cell invasion by processing a variety of substrates including matrix metalloproteinases (MMPs). PACE4, a member of the family of PCs was shown to enhance mouse skin carcinoma progression by increasing tumor cell invasiveness. However, the effects of PACE4 on malignant conversion have not been investigated. In the present study we address the possible role of PACE4 as a trigger of malignant conversion by transfecting with a full-length PACE4 cDNA, three keratinocyte cell lines with no or little tumorigenic potential, i.e. non-tumorigenic BALB/MK-2 cells, tumorigenic non-invasive MT1/2 cells and tumorigenic moderately invasive p117 mouse skin keratinocytes. Overexpression of PACE4 led to a significant increase in the processing of stromelysin-3, a well-characterized substrate of this PC. When assayed for invasive ability, the PACE4-transfected cells were invasive both in vitro and in vivo, whereas their control counterparts were not. In addition, an enhanced processing ability of MT2-MMP a known substrate of PCs was detected in the PACE4-transfected cells. This was accompanied by MMP-2 and MMP-9 activation in PACE4 transfectants. Invasion and MMP processing were remarkably reduced when PACE4 was inhibited with a specific antibody. By triggering the processing of crucial invasion-related proteases, PACE4 is not only able to enhance the invasive ability of malignant cells as demonstrated previously, but also played a significant role in converting non-invasive keratinocytes into malignant cells.

Abbreviations: Ab, antibody; DD, differential display; ECM, extracellular matrix; FBS, fetal bovine serum; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; Pen-Strep, penicillin-streptomycin; PC, proprotein convertase; Sc, subcutaneous; SCC, squamous cell carcinoma; SPCC, spindle cell carcinoma; Str3, stromelysin-3


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many proteins are made as inactive precursors that are later processed by proprotein convertases (PCs) to yield biologically active proteins. The subtilisin-like serine proteases are a family of PCs involved in this processing. All PCs share structural similarities and are involved in the processing of protein precursors such as hormones, adhesion molecules, growth factors, receptors, matrix metalloproteinases and plasma proteinases. Cleavage occurs at single basic or paired basic residues of the proproteins (1–4). To date seven members of the PCs have been identified which include PC1/PC3, PC2, PACE4, PC4, PC5/PC6, PC7/PC8/LPC and furin (1,5–11). PACE4 was first cloned from a human hepatoma cDNA library (11). Its location and proximity to the fur gene (11) suggest that they might have evolved from the same gene. PACE4 displays the highest expression in the anterior pituitary (12,13) but also has been reported to be expressed to some extent in many other tissues (4,11,14). Many PCs are overexpressed in various types of cancer, such as lung (15,16), breast (17) and skin (18). Some of the PC-activated substrates have significant roles in cancer development. Among these substrates, metalloproteinases, growth factors and adhesion molecules are directly related to tumor progression by either modulating the degradation of the extracellular matrix (ECM), or by influencing cell adhesion/locomotion and growth.

Late, during tumor development, tumor cells acquire the ability to degrade the ECM and invade surrounding tissues. Stromelysin-3 (Str3) is a metalloproteinase (MMP), one of the enzyme family involved in degrading the components of ECM, and therefore involved in tumor invasion. Str3 has an insertion of 10 amino acids between the propeptide and the catalytic domain, including the paired basic amino acid recognition site for convertase processing enzymes and can be processed by both furin and PACE4 (19,20). Membrane-type MMPs (MT-MMPs), e.g. MT1-, MT2- and MT3-MMPs (21) also have the recognition site for serine proteases and upon activation can process pro-MMP-2 (22–24). Once activated, MMP-2 degrades collagen IV, one of the major components of the basement membrane. Overexpression of MT-MMPs has been observed in many cancers including head and neck (25), skin (26), pancreatic (27) and lung (22) cancers. Moreover, the levels of expression of these MT-MMPs indicate the degree of malignancy of the tumors.

Our previous studies of mouse skin carcinoma cell lines, showed differential expression of PACE4. PACE4 was overexpressed in some cell lines characterized by a more aggressive behavior and spindle cell morphology, whereas malignant cell lines with a more differentiated squamous phenotype had little or no expression of PACE4 (18). In addition, transfection of a tumorigenic squamous cell carcinoma (SCC) cell line with the PACE4 gene converted the SCC cell into a more invasive variant (18). PACE4 is also overexpressed in human cancers, e.g. lung and breast at invasive/metastatic stages (16,17). All studies on PACE4 describe the role of this PC at late invasive/metastatic stage of tumor progression. There is no study showing whether PACE4 overexpression at early stages of tumorigenesis can contribute to malignant conversion of the cells. In addition, the biochemistry of PACE4, e.g. its physiological substrates, requires more characterization. To better understand at which stage of tumorigenesis PACE4 expression is sufficient to render tumor cells invasive and to determine the mechanism of action of this PC, we analyzed the invasive ability, PACE4 activity, MMP processing and gelatinolytic activity of non-tumorigenic and papilloma-derived cell lines transfected with PACE4 cDNA.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines
The mouse skin keratinocyte cell lines C50, BALB/MK-2, MT1/2, p117, VT17DT and HEL30 (28–33) were used to screen for the endogenous expression of PACE4. C50 was supplied by Dr A.Balmain (Beatson Inst., Glasgow, UK) and the other cells by Dr Susan Fischer (MDACC Science Park, Smithville, TX). C50 and BALB/MK-2 are immortalized normal keratinocyte cell lines, the remaining cell lines are derived from benign skin papillomas. hEK293.P4, supplied by Dr Richard Mains (University of Connecticut, Farmington, CT), are human kidney cells stably transfected with the PACE4 gene, and were used as a positive control for PACE4 expression. All cells were grown in SMEM medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine and penicillin-streptomycin (Pen-Strep) (100 U/ml and 100 mg/ml, respectively).

Transfection of mammalian cell lines
BALB/MK-2, MT1/2 and p117 cells were transfected with either pCI.neo (Promega, Madison, WI, mammalian expression vector) or pCI.neo.PACE4 plasmid (full-length PACE4 cDNA cloned into the EcoRI–SalI site of pci.Neo plasmid) (18). Transfections were performed according to manufacturer's instruction (Lipofectamine Plus Reagent; Gibco BRL, Grand Island, NY). Transfected cells were grown in SMEM, 10% FBS, L-glutamine and Pen-Strep, and selected in gentamicin (G418, 800 mg/ml) 2 days after transfection. Cells were propagated and single clones were selected. Expression of PACE4 was evaluated by western blot and PACE4 activity of cells was determined by pro-stromelysin-3 (pro-Str3) processing, as described below. Cells with high PACE4 expression and activity (Str3 assay) were chosen for further studies.

Western blots
Detection of PACE4.
Cells were starved overnight with SMEM medium containing L-glutamine and Pen-Strep (serum-free medium). Conditioned media of selected single clones were collected after 16 h of incubation and concentrated using Amicon centripreps (Fisher, Springfield, NJ). Equal amounts of total proteins were electrophoresed through 8% denaturing Novex pre-cast polyacrylamide gels (Invitrogen/Novex, Carlsbad, CA) and transferred to nitrocellulose membrane. The primary antibody was JH1475, a rabbit polyclonal antibody against amino acids 570–656 of PACE4 (12). The secondary antibody was anti-rabbit linked horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was washed and developed using Amersham ECL chemiluminescence reagent.

Detection of MMP-2.
The detection of MMP-2 was similar to the PACE4 detection with the exception that cells were incubated for 24 h with either pre-immune rabbit serum or with the PACE4 antiserum (1:500 dilution) prior to the starvation and the starvation was performed overnight with or without the antibody. Primary antibody was goat polyclonal MMP-2 antibody (catalog no. sc-6838; Santa Cruz Biotech, Santa Cruz, CA). The secondary antibody was anti-goat linked (Santa Cruz Biotech) horseradish peroxidase.

Detection of MT2-MMP.
Cells were grown overnight in a 24 well plate at a concentration of 105/well, with either pre-immune rabbit serum or with the PACE4 antiserum (1:500 dilution). Cells were then washed with 1x PBS and lyzed in RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (PMSF, sodium orthovanadate and aprotinin) at 4°C for 1 h. Cell lysates containing the same amounts of protein were run through a 10% denaturing gel as explained before. A monoclonal antibody was used as the primary antibody (catalog no. MAB3320; Chemicon International, Temecula, CA), and anti-mouse linked horseradish peroxidase (Amersham Pharmacia Biotech) was used as the secondary antibody.

St3 assay.
Cells (3x104) were grown overnight in a 24 well plate in SMEM containing 10% FBS, L-glutamine and Pen-Strep. The medium of each cell line was replaced by the assay mix (140 ml NIH3T3 conditioned medium, 20 ml of 10 mM CaCl2, 40 ml of Str3 containing medium; 34) and incubated overnight. Negative control is the assay mix incubated overnight at 37°C with no cells. The assay mix was collected and electrophoresed through an 8% Tris–glycine gel (Invitrogen/Novex) under denaturing conditions. Proteins were transferred to membrane and detected as described before. Primary antibody was 5ST-4A9, a monoclonal antibody raised in mouse against Str3 (a gift of Paul Basset's laboratory, Strasbourg, France).

Doubling time
Cells were grown in 12 well plates and counted every other day. Log of cell numbers versus days was plotted to obtain the doubling time for each transfected cell line.

In vitro invasion assay
In vitro invasion assay of transfectants was performed using BD Biocoat Matrigel Invasion Chambers (Becton Dickinson, Bedford, MA), according to manufacturer's instruction, in presence or absence of PACE4 antiserum. To assess the invasive ability of cells in the presence of PACE4 antibody, cells were incubated with either PACE4 antiserum or with its respective pre-immune rabbit serum 24 h prior to and during the invasion assay at 1:1000 and 1:500 dilutions.

In vivo invasion assay
Tracheal transplants were prepared as described previously (35,36). Cells (5x105) of each cell line were inoculated into de-epithelialized rat trachea (Zivic-Miller, Pittsburgh, PA). Six to 10 tracheas were used for each cell line. After inoculation of cells into tracheas, tracheas were sealed and transplanted into the dorsal subcutaneous tissues of SCID mice. Tracheal transplants were removed surgically at 4–6 weeks, sectioned into 3 mm thick rings and fixed in 10% formalin. Following hematoxylin and eosin staining, the degree of invasion of the tracheal wall was determined semiquantitively based on the level of penetration of transfectant cells. Level 0, cells did not grow in the trachea. Level 1, no invasion of the wall, cells confined to the lumen or lining the luminal surface. Level 2, tumor cells found in the mucosa and superficial lamina propria. Level 3, the lamina propria completely infiltrated by tumor cells and the pars membranacea and trachealis muscle invaded. The adventitia is not invaded by tumor cells. Level 4, the malignant cells have reached the adventitia, and the whole tracheal wall is invaded but the invasive tumor does not penetrate >1 mm outside the tracheal wall. Level 5, massive invasion of the tracheal wall with penetration of the invasive cells >1 mm outside the xenotransplanted organ.

Zymography
Cells (1x106) were grown overnight in a serum-free SMEM medium containing L-glutamine and Pen-Strep. The conditioned media were concentrated down to 200 ml using Amicon centripreps (Fisher, Springfield, NJ) and 20 ml of each sample was loaded on a 10% Novex precast zymogram (gelatin) gel. The gel was run, renatured and developed according to the manufacturer's instruction. Gelatinase Zymography Standards was purchased from Chemicon International (Temecula, CA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of endogenous PACE4 in cell lines
Potential candidates for transfection were screened for endogenous PACE4 expression. Previous studies on the tumorigenicity of cells revealed that spontaneously immortalized, epidermal keratinocytes C50 and BALB/MK-2 cell lines are non-tumorigenic (37), papilloma-derived MT1/2 is tumorigenic but non-invasive (33) and papilloma-derived p117, VT17DT, HEL30 are tumorigenic and slightly invasive (33). All cells lines had remarkably low expression of PACE4 (Figure 1AGo) and hence could be recipient of PACE4 cDNA. One cell line from each category was chosen for transfection, those included BALB/MK-2 (non-tumorigenic), MT1/2 (tumorigenic, non-invasive) and p117 (tumorigenic and slightly invasive).



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Fig. 1. (A) Western blot of PACE4 in mouse skin cell lines. Conditioned media containing 50 mg of total protein were loaded on a 8% SDS–PAGE. Conditioned medium of hEK293.P4 containing 10 mg of protein was used as positive control. Detection was performed using the polyclonal antibody JH1475. (B) Western blot analysis of cell lines transfected with either pCI.neo (vector alone) or with PACE4 cDNA. Conditioned medium of transfected cells containing 20 mg of total protein were loaded on a 8% SDS–PAGE, detection was performed as in (A). After screening of clones, the above transfected-cells were selected for further studies. (C) Str3 activation assay (western blot). Conditioned medium of MCF-7 cells containing pro-str3 protein was incubated with each of the transfected cell lines overnight. The assay mix was loaded on 8% denaturing gel. Detection was performed using the monoclonal antibody 5ST-4A9 against Str3. The processed Str3 is ~49 kDa and is marked with an arrow. The solid line represents pro-Str3.

 
Transfection of cell lines with PACE4 cDNA
To examine how PACE4 might influence the properties of benign cells, BALB/MK-2, MT1/2 and p117 cell lines were transfected with either vector pCI.neo (hereafter called cin) or with vector containing PACE4 cDNA (P4) creating three pairs of transfected cells. Single clones of each pair of transfected cells were screened by western blot, and one clone per cell line was selected for further studies (Figure 1BGo). As shown in Figure 1Go, BMK.P4, MT.P4 and p117.P4 have higher PACE4 expression than their vector alone counterparts, BMK.cin, MT.cin and p117.cin, respectively. The highest level of PACE4 expression is observed in PACE4-transfected p117 cells and the lowest in PACE4-transfected MT1/2 cells. Doubling time indicated that there were no significant growth differences between the vector alone and the PACE4-transfected cells (data not shown). These pairs of cells were employed for the analysis of MMP processing and in vitro and in vivo invasiveness.

PACE4 activity of transfectants
Pro-Str3 was shown previously to be a PACE4 substrate (17). To confirm that PACE4-transfected cells exhibited PACE4 activity, an assay was performed to assess Str3 processing. Conditioned medium containing pro-Str3 (gift of Paul Basset's laboratory) was used as the source of substrate for PACE4 (34). Cells were incubated overnight with assay mix containing pro-Str3. Western blot analysis of the medium revealed that for each pair of cell lines, the PACE4-transfected cells exhibited enhanced ability to process Str3 into its mature form of lower molecular weight (Figure 1CGo, arrow) than the vector-alone-transfected cells.

MT1- and MT2-MMP have the consensus sequence for PC-cleavage (21). Although MT1-MMP is shown to be cleaved by furin-like proteases (21), activation schemes for MT2-MMP have not been delineated to date. However, given the presence of similar basic recognition motifs in homologous regions of their prodomains (21), MT1- and MT2-MMPs are likely processed to their mature form via similar mechanisms. Once activated, MT-MMPs can in turn activate MMP-2 (19,21,38). Given the potency of MT-MMPs to be processed by PCs and to activate MMP-2, processing of MT1-MMP, MT2-MMP and MMP-2 was evaluated in the transfected cells. MT1-MMP showed little expression and thus no evidence of processing (data not shown). MT2-MMP was processed in all three PACE4-transfected cells (Figure 2AGo). Processing was more prevalent in p117.P4 and BMK.P4 cells than MT.P4 cell line. This was also true for MMP-2 processing (Figure 2CGo). When PACE4 was inhibited with the rabbit PACE4 anti-serum, activation of both MT2-MMP (Figure 2AGo) and MMP-2 (Figure 2CGo) were greatly reduced in the PACE4 overexpressing cells. The pre-immune serum had no inhibitory effect on activation.



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Fig. 2. Activation of MMPs. Solid lines mark the pro- and arrows mark the processed MMPs. (A) Western blot of MT2-MMP. Cells were grown in 24 well plates (see Materials and methods) and lyzed for an 1 h. Cell lysates were loaded on a 10% gel. MT2-MMP was detected with monoclonal antibody MAB3320. (B) Actin western blot of (A) to monitor for equal loading. (C) Western blot of MMP-2. Conditioned medium of each cell line was concentrated by ultrafiltration and loaded on an 8% gel. MMP-2 was detected with polyclonal antibody sc-6838. P4 expression + indicates expression of PACE4-transfected cells. P4 expression – indicates no expression in vector alone-transfected cells. P4 antibody + indicates the use of the anti-PACE4 serum in the medium, whereas P4 antibody – indicates the use of the pre-immune serum (both at 1/500 dilution). (D) Gelatin zymogram of the transfected cell lines. Conditioned media of transfected cells were concentrated by ultrafiltration and loaded on a 10% zymogram gel. Note the slight processing of MMP-2 into the intermediate form in the p117.cin cell line. Activation of MMPs by MT.cin.P4 cells is less efficient than their activation by the other two PACE4-transfected cells.

 
MMP-2 and MMP-9, an MMP-2 substrate, are involved in degradation of collagen IV one of the major components of the ECM. Gelatin zymography was employed as a tool to evaluate the ability of the transfectants to degrade collagen IV. As depicted in Figure 2DGo, PACE4-transfected cells exhibit increased activation of MMP-2 as compared with the control cells. The increased MMP-9 gelatinolytic activity was more prevalent in BMK.P4 and p117.P4 than in MT.P4 cells.

Invasiveness of transfected cells
To study whether PACE4 overexpression can affect the invasive behavior of the cells, in vitro and in vivo invasion assays were performed. The in vitro assay evaluated the invasive ability of BMK- and p117-transfectants by counting percentage of cells that degraded and passed through the Matrigel inserts. In the absence of antibody (pre-immune serum), BMK.P4 exhibited a 3-fold increase and p117.P4, a 2–3-fold increase in their invasiveness (Figure 3A and BGo) compared with the control vector-transfected cells (Figure 3A and BGo). The invasive behavior of BMK- and p117 PACE4-transfectants was significantly reduced when PACE4 antiserum was added to the invasion chambers (P < 0.05) (Figure 3A and BGo). As depicted in Figure 3Go, the effect of PACE4 antiserum is dose-dependent. The invasive behavior of vector-alone-transfected cells was low and did not change significantly in the presence of PACE4 antiserum (Figure 3A and BGo). MT-transfectants did not survive the serum-free condition required for the in vitro assay.



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Fig. 3. In vitro invasion assay of BMK- (A) and p117-transfectants (B). Cells were grown in the invasion chambers for 24 h with either pre-immune serum (PI) or with anti-PACE4 serum (two dilutions 1/1000 and 1/500). Cells that passed through the Matrigel covered porous filter were stained and counted, and an average of 10 random fields/slide was counted. Experiments were done in triplicate, and standard deviations of the three separate experiments were calculated.

 
In the in vivo invasion assay (tracheal xenografts) each selected cell line was introduced into de-epithelialized rat tracheas and inoculated into the dorsal subcutaneous region of SCID mice for a period of 4–6 weeks. The degree of invasion of the tracheal wall was determined semiquantitatively based on the level of penetration of transfected cells (Figure 4AGo). Figures 4B–G and 5A–FGoGo show examples of stained cross sections of tracheal transplants at different magnifications. Figure 6Go summarizes the invasion levels for each pair of cell line.



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Fig. 4. In vivo invasion assay. (A) Levels of invasion after xenotransplantation of cultured cells into deepithelialized tracheal transplants. Level 0, no tumor cell growth; level 1, tumor cells lining the tracheal luminal surface; level 2, partial or total obliteration of lumen by tumor cells; level 3, pars membranacea is invaded; level 4, entire tracheal wall, including adventitia, is involved; level 5, (<1m m) adventitia and surrounding tissues (>1 mm) are involved. (B–G) Cross sections of tracheal containing either PACE4- or vector-alone-transfected cells. The BMK.cin cells grew forming a single layer of non-invasive cells that covered the lumen (B). The BMK.cin.P4 cells showed an invasive behavior growing into the lamina propria and the pars membranacea (arrow) (C). The vector-alone-transfected MT.cin cells could be seen growing as a single layer of flat cells that did not penetrate into the tracheal wall (D). The PACE4-transfected MT.cin.P4 cells formed a thicker layer of cells that pushed into the underlying subepithelial tissue where a clear desmoplastic reaction can be noted (arrow) (E). p117.cin cells (F) and p117.cin P4 cells (G) were both invasive and penetrated deep into the tracheal wall. Nevertheless, the PACE4-transfected cells grew faster and deeper into the adjacent tissues (arrow) than the p117.cin cells. Hematoxylin and eosin x8.5.

 


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Fig. 5. Histopathological detail of the xenotransplanted cells growing in tracheal transplants. The vector-alone-transfected BMK.cin cells cover the luminal surface forming a flat monolayer of typical non-invasive epithelial cells (arrow) (A). The corresponding PACE4-transfected cells form large groups of atypical invasive squamous carcinoma cells (B). MT.cin cells likewise form a simple flat epithelium (arrow) that does not invade (C) and the PACE4-transfected MT.cin.P4 cells formed a thick epithelium with hyperkeratosis and dysplastic cells (arrowheads). In the subepithelial tissue invading carcinomatous structures can be observed (arrows) (D). Both p117.cin (E) and p117.cin.P4 (F) cells invaded the tracheal wall as moderately to poorly differentiated squamous cell carcinoma cells. Note that the latter cells extend deeper into the adjacent tissues and that the center of the trachea becomes necrotic. Hematoxylin and eosin, x85.

 


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Fig. 6. Summary of in vivo invasion assay for transfected cell lines. Trachea were removed from mice at 4–6 weeks of inoculation, stained and levels of invasion (described in Figure 4AGo) were determined. Two sided P-values are 0.004, 0.006 and 0.025 for BMK-series, MT-series and p117-series, respectively.

 
The in vivo invasion assay of the BALB/MK-2 series revealed that the PACE4 expressing cells were more invasive than the control (vector-alone-transfected) cells. BMK.cin, the vector-alone-transfected cells, constituted a flattened or cuboidal simple (one layer) epithelium that covered the luminal surface without penetrating the subepithelial tissues (Figures 4B and 5AGoGo). Transplanted BMK.cin.P4 cells were different from the vector-alone-transfected cells. These cells formed epithelial nests and masses that penetrated into the pars membranacea and the adventitia (Figures 4C and 5BGoGo, stages 4–5). Figure 6Go shows that most tracheas containing BMK.P4 cells reached invasion levels of 2 and higher, while the vector-alone-transfected cells remained mostly at level 1.

Transplantation of MT.cin cells (transfected with vector alone), showed that in most tracheal transplants (seven of 11; Figure 6Go), the cells grew in the tracheas limiting themselves to cover the tracheal lumina without penetrating into the subepithelial tissues (Figures 4D and 5CGoGo, stage 1). Four tracheas inoculated with MT.cin cells did not show any growth whatsoever (Figure 6Go, stage 0). The MT.P4 cells (PACE4-transfected) were able to invade the subepithelial tissues, some of them invading the pars membranacea (Figure 4EGo, stage 3) and eventually reaching the adventitia (stage 4) in 50% of the xenografts (Figure 6Go). The other half remained in stage 1 without obvious invasion. However, there was a remarkable difference between MT.cin cells at stage one and MT.cin.P4 cells at the same numerical stage. MT.cin cells always remained inside the tracheal cavity space and lined the lumen forming an indolent-looking simple or bistratified epithelium with no or minimal keratinization. In contrast, MT.cin.P4 (the PACE4-transfected cells) were larger with more prominent nuclei and nucleoli and formed a multi-layered stratified epithelium with marked keratinization that accumulated in the lumen of the transplant. Furthermore, the cells lining the lumen seemed to push towards the outer layers of the tracheal wall (Figure 4EGo), even in those cases that were classified as stage 1. The epithelium that covered the tracheal lumina was stratified with marked nuclear atypia and superficial keratinization. Very clear invasion of the subepithelial tissues could be seen at higher magnification in half of the transplants (Figures 5D and 6GoGo).

Both vector-alone and PACE4-p117-transfected cells were invasive (Figures 4F–G, and 5E–FGoGo). Nevertheless, p117.P4 cells were more invasive. In four of six tracheal transplants p117.P4 cells reached stage 5 after 6 weeks. Most tracheal grafts containing p117.cin cells only reached stages 3 and 4 at the same time point (Figure 6Go).


    Discussion
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In a previous study differential display was used to identify genes that mark essential steps in the conversion of a squamous cell carcinoma (SCC) to a more aggressive, poorly differentiated spindle cell tumor (SPCC). This resulted in the identification of PACE4 overexpression in the aggressive SPCCs and low or absent expression in the more differentiated and less invasive SCCs (18). The experiments indicated that PACE4 could be a significant factor driving skin tumor progression.

Tumor progression is a stepwise process in which non-tumorigenic cells change from benign/non-invasive to more invasive, and highly invasive/metastatic variants. This progression involves multiple genetic alterations. In the present studies we investigated the effect of ectopic PACE4 expression using cells that have no or minimal neoplastic features, e.g. non-tumorigenic immortalized keratinocytes and papilloma-derived cell lines. To our knowledge this is the first report showing that transfection of non-tumorigenic cell lines with a PC, in this case PACE4, is sufficient to confer an invasive phenotype. We also demonstrate for the first time that MT2-MMP could be the physiological substrate of PACE4 under conditions that result in overexpression of this protease. PACE4 overexpression led to enhanced MT2-MMP, MMP-2 and MMP-9 processing. As expected from activation of the two latter MMPs, PACE4 overexpressing cells, e.g. BMK.P4, MT.P4 and p117.P4 cells were significantly more invasive than their vector-transfected counterparts. In the p117-transfectants, the vector-transfected cell line is also invasive. This could be explained by the finding that vector-alone-transfected p117 cells also processed, to some degree, Str3, MT2-MMP and MMP-2. The in vitro invasion of p117.cin cells was not significantly affected by the increasing concentration of PACE4 antiserum. Nevertheless, MT2-MMP and MMP-2 were processed to some extent. This points to the presence of PCs other than PACE4 capable of processing these substrates in the p117 parental cell.

Activation of MT-MMPs by PACE4 is not unexpected. The zymogens of MT1-, MT2- and MT3-MMPs contain a consensus cleavage site for at least four members of the PC family, i.e. furin, PC6, PC7 and PACE4 (1,21,39). Although furin has been shown to efficiently activate MT1-MMP (40), endogenous PCs, such as PC7 and PACE4, partially processed MT1-MMP in human cell lines (41). Overexpression of MT1-MMP has been extensively described in human malignancies (42,43). Expression of MT2-MMP was found to be very high and more frequent than MT1-MMP in ovarian carcinoma effusions (38). Furthermore, the expression levels of both MT1-MMP and MT2-MMP were enhanced in pancreatic cancer (24). Among the MT-MMPs, MT1-, MT2- and MT3-MMPs share common substrates and are able to process MMP-2 (21–23,44). In our murine cell system MT2-MMP was efficiently activated in PACE4-transfected cells. Activation of MMP-2 by MT-MMPs is predominately observed in invasive tumor cells (45) and is followed by proteolytic degradation of type IV collagen. The active MMP-2 can also process MMP-9 (46) another member of the MMPs involved in degradation of type IV collagen. RXK/RR sequence is known to be the cleavage site targeted by most of the members of the subtilisin-like serine proteases, including furin and PACE4. PACE4 and furin share common substrates (47) and can be inhibited by common inhibitors (48,49), i.e. PACE4, furin, PC6 and PC7 are shown to process pro-IGF-2 at Arg 104 to generate active IGF-2 (50) which is involved in tumor progression (51). The furin-null CHO-k1 cells, unable to process their insulin pro-receptor, had partial rescue of the mutant phenotype after being transfected with human PACE4 (52). Therefore, furin substrates could be potential substrates for PACE4. For this reason, transfectants were screened for processing of MT1-MMP, transforming growth factor TGF-ß (53), insulin-like growth factor-2 (IGF-2) (50) and insulin-like growth factor receptor (IGF-1R) (54). The above substrates were either not detected (TGF-ß and IGF-2) (data not shown), or were detected at very low levels and did not seem to contribute to the phenotypic changes of the transfected cells (MT1-MMP and IGF-1R) (data not shown). In our system, the only substrate that was detected and its processing conclusively contributed to the described phenotypic changes was MT2-MMP.

PACE4 was shown previously to be overexpressed in various types of cancer. When the levels of convertases were examined in human lung neoplasms, PACE4 transcripts were detected in eight of 14 adenocarcinomas, seven of 17 squamous cell carcinomas and two of seven small-cell lung carcinomas (SCLCs) (16). PACE4 mRNA was detected in seven of seven human breast cancer cell lines and 30 of 30 breast tumor tissues studied by Cheng et al. (17). In addition, when PACE4 expression in primary mouse spindle cell tumors were examined (18) ~50% of the SPCCs studied (seven of 15) had moderate to high levels of PACE4 expression. The above findings indicate the possible role of PACE4 in tumor progression and invasion. The present studies have determined that PACE4 is able to induce malignant conversion at early stages of tumorigenesis, e.g. in non-tumorigenic BALB/MK-2 and in tumorigenic non-invasive MT1/2 cells.

It is quite remarkable that transfection of a single gene such as PACE4 results in considerable phenotypic changes. Nevertheless, this is not the only example of such a phenomenon. Transfection of cell lines with a single gene was shown by different groups to modulate cell invasion and metastasis. Such examples include transfection of cells with MEK1 (55), N-cadherin (56), bcl-2, (57), AAC-11 (58) and H-ras (59). Most of the above examples induce invasion and metastasis, at least in part, through increased expression and/or activation of MMPs, especially MMP-2 and MMP-9. In addition, several reports have described the direct transfection of MMP genes (25,45,60–64), including transfection of MT2-MMP, that resulted in an enhanced malignant or malignant-like phenotype, including increased motility, invasiveness and metastasis.

Taken together, PACE4-transfected cell lines derived from either immortalized non-tumorigenic or papilloma-derived keratinocytes showed a remarkable invasiveness when compared to the respective vector-alone-transfected counterparts. The increased invasive behavior of PACE4-transfected cells correlated with their ability to process Str3 and MT2-MMP. The reduced invasion of PACE4-transfected cells in the presence of PACE4 antibody additionally proves that the invasive property of these cells is PACE4-dependent. The results clearly show that overexpression of PACE4 under conditions that simulate the in vivo milieu, can lead to a remarkable change of transfected cells, due to the direct or indirect activation of MMPs.


    Notes
 
1 To whom correspondence should be addressed Email: aj_klein-szanto{at}fccc.edu Back


    Acknowledgments
 
We thank Drs R.Mains for providing antibody and PACE4 cDNA, S.Fischer and A.Balmain for providing mouse skin cell lines, the P.Basset's laboratory for providing Str3 antibody, and G.Kruh for helpful discussion. This work was supported by NIH-NCI grants CA 75028, CA 06927, and by an appropriation of the Commonwealth of Pennsylvania.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 6, 2001; revised January 8, 2002; accepted January 14, 2002.