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1 School of Life Sciences and Science Research Centre, Queensland University of Technology, Brisbane, Australia
2 Wesley Research Institute, Brisbane, Australia
3 Department of Urology, Princess Alexandra Hospital, Brisbane, Australia
(Requests for offprints should be addressed to J Clements, Prostate Cancer Research Program, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, 4001, Australia; Email: j.clements{at}qut.edu.au)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Epithelial-mesenchymal transition (EMT) is a crucial event in the progression of cancer to an invasive phenotype. The perturbation of E-cadherin and increased expression of vimentin are two currently accepted characteristics associated with EMT. E-cadherin is downregulated in most epithelial cancers, and can be correlated to higher mobility and invasiveness of tumour cells (Hirohashi & Kanai 2003). The change in E-cadherin expression observed in many prostate cancer cases can be attributed to methylation of the E-cadherin promoter (Kallakury et al. 2001) or truncation of the protein leading to its inactivation (Davies et al. 2001). In contrast, vimentin expression is usually increased in cells which have undergone EMT and has been characterised in migrating cancer cells (Hendrix et al. 1997, Gilles et al. 1999, Singh et al. 2003). Vimentin is associated with increased in vitro motility of prostate cancer cell lines, and immunohistochemical studies have shown elevated levels of vimentin in the majority of advanced prostate cancer tissues and bone metastases (Lang et al. 2002).
In this study, we sought to determine the relative expression levels of hK4, compared with the more well-known PSA and hK2, and to determine its involvement in biological processes associated with cancer progression. We have unequivocally shown, for the first time, a clearly increased expression of hK4, at the protein level, in cancer compared with benign tissues. This observation not only shows that hK4 is more cancer specific in expression than PSA or hK2 – thus having potential as a useful diagnostic/prognostic biomarker for prostate cancer – but also suggests that hK4 may play an important functional role in prostate cancer progression. To elucidate such a role, we have characterised PC-3 cells transfected separately with the three prostate-specific kallikreins, KLK4, KLK3/PSA and KLK2. We report that the expression of hK4 and PSA, but not hK2, promotes cell migration and alters the structure of these cells. E-cadherin expression is lost, while vimentin expression is increased, both hallmarks of EMT-like behaviour. These findings suggest that the prostatic kallikreins, hK4 and PSA, may play a role in the initiation of EMT-like changes in prostate cancer cells and thus are important modulators of prostate cancer progression.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Formalin-fixed paraffin blocks from prostate tumours (n=6) and benign prostatic hyperplasia (BPH) (n=6) were sectioned (4 µm), deparaffinised and rehydrated. After H2O2 treatment to quench endogenous peroxidase, the sections were incubated overnight with an anti-PSA polyclonal antibody (1:5000 dilution; Dako, Botany, NSW, Australia), anti-hK2 monoclonal antibody (1:700 dilution; clone HK1G 586.1; Hybritech, San Diego, CA, USA), or anti-hK4 polyclonal antibody (1:250 dilution) at 4 °C respectively. The EnVision+ peroxidase polymer detection system (Dako) was used with 3,3'-diaminobenzidine (DAB; Sigma, Castle Hill, NSW, Australia) as the chromogen. The sections were counterstained with Mayer’s haematoxylin. Normal goat serum (10%) replaced the primary antibodies as a negative control. The hK4 peptide antibody was generated by immunisation of New Zealand rabbits using a peptide, IIN-GEDCSPHSQ, designed to the NH2-terminal region of hK4. The production, specificity and characterisation of the hK4 antibody is described elsewhere (Harvey et al. 2003).
Cell lines, expression plasmids and transfection
PC-3 and LNCaP prostate cancer cell lines (American Type Culture Collection, Rockville, MD, USA), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 units/ml penicillin G sodium and 100 µg/ml streptomycin sulphate. For transfection, the entire coding region (pre-pro-enzyme) of KLK4, KLK3 or KLK2 cDNA was amplified from mRNA extracted from LNCaP cells and inserted into the mammalian expression vector, pcDNA3.1 (Invitrogen, Mt Waverley, Victoria, Australia). PC-3 cells, which do not express KLK3 or KLK2 and express low levels of KLK4 endogenously, were transfected with the individual plasmids (pcDNA3.1:KLK4, pcDNA3.1:KLK3 or pcDNA3.1:KLK2) or an empty vector plasmid (pcDNA3.1:vector only), using the lipid-mediated Lipofectamine 2000 Reagent protocol (Invitrogen). Stably transfected cells were selected in medium containing 100 µg/ml G418 (Invitrogen) and are denoted as PC-3:hK4 (nos. 1–6), PC-3:PSA (nos. 1–7), PC-3:hK2 (nos. 1–7) or PC-3:vector only (nos. 1 and 2) cell lines.
Western blot analysis
Western blotting was performed with cell lysates collected from several cell clones, while conditioned medium was collected after serum starvation for 48 h and concentrated 20-fold. Total protein concentrations were measured by the micro-bicinchoninic assay (BCA; Pierce, Progen, Darra, Queensland, Australia) with BSA standards. An amount of 10 µg protein samples was separated on 8–12% SDS polyacrylamide gels under denaturing conditions, transferred onto nitrocellulose membranes (Schleicher and Schuell; Medos, Mount Waverley, Victoria, Australia) and stained with Ponceau S (Sigma) to ensure that transfer and equal loading had occurred. Membranes were quenched with 5% skim milk blocking solution before primary antibodies were added and incubated at 4 °C overnight. Primary antibodies were as follows: hK2–HK1G 586.1 (Hybritech, San Diego, CA, USA), PSA–PSM 773.3.3 (Hybritech) and hK4–NH2-terminal peptide antibody. Horseradish peroxidase-conjugated goat anti-mouse (hK2 and PSA) or goat antirabbit (hK4) secondary antibodies (Pierce) were applied before the addition of West Femto substrate (Pierce), chemiluminescent exposure to radiographic film and development with a Curix 60 automatic developer (Agfa, Stafford, Queensland, Australia).
RT-PCR and real-time PCR
Total RNA was extracted with TRI-Reagent (Sigma). cDNA was synthesised using Superscript II (Invitrogen), and RT-PCR and real-time PCR were performed with primers specific to the gene of interest (see figure legends). Real-time PCR was performed on an ABI 7000 Thermal Cycler using the SYBR Green I Dye detection system (Applied Biosystems, Scoresby, Victoria, Australia). Relative levels of gene expression were normalised to 18S ribosomal RNA. Each sample was amplified three times in triplicate. Statistical analysis was performed with Student’s t-test.
Immunofluorescence and confocal microscopy
Cells were grown on sterile 8- or 16-well glass chamber slides (Medos). Double labelling for actin (tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin) and vinculin (clone 7F9) was performed with an actin cytoskeleton and focal adhesion staining kit (Chemicon, Boronia, Victoria, Australia). Tubulin was detected with the clone 2G10 antibody (Upstate Biotechnology, Auspep, Parkville, Victoria, Australia). Antibodies against E-cadherin (HECD-1 (extracellular domain) and 4A2C7 (intracellular domain); Zymed, Gymea, NSW, Australia) and vimentin (LN6; Sigma) were used for immunofluorescence. Secondary incubations were performed with AlexaFluor 488 goat antimouse immunoglobulin (Ig)G antibody (Molecular Probes, Bioscientific, Gymea, NSW, Australia). A Leitz fluorescent compound microscope and/or a Leica TCS 4D confocal microscope were used for imaging.
MTT cell proliferation assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assays were based on a reported method (Mosmann 1983), with modifications. All cell line clones were used in this assay and assessed every 24 h for a total of 96 h. Experiments were assayed with 18–24 replicates and repeated three times. Statistical analysis was determined at 96 h by Student’s t-test.
In vitro motility and Matrigel invasion assays
Cell motility was determined by a reported procedure (Saito et al. 1997). PC-3 cell lines (5 x 104) were seeded onto 8 µm pore tissue culture inserts (Falcon, BD Biosciences, North Ryde, NSW, Australia) in serum-free medium containing 0.1% BSA, and allowed to migrate toward the chemoattractant (20% FCS) for 24 h (hK4-expressing cells) or 48 h (PSA- and hK2-expressing cells). Invasion assays were performed as for the motility assays but with growth factor-reduced Matrigel (BD Biosciences) coated on the upper surface of the insert. All assays were performed in duplicate on four occasions, with proliferation over the experimental period taken into account. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc analysis.
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Results |
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To validate the previous reports of increased hK4 mRNA in prostate cancer, we first determined the relative expression levels of hK4 (protein) compared with PSA and hK2 in cancer and benign glands. This was done by immunohistochemistry, and the results shown in Fig. 1
(A–F) are representative of the staining patterns observed for all BPH (n=6) and prostate cancer (n=6) tissue sections. As expected, hK4, PSA and hK2 expression was primarily localised to the cytoplasm of epithelial cells in both benign and cancer glands. Weak staining was observed in the epithelium of the benign glands stained with the hK4 peptide antibody, whereas strong immunoreactivity was found in the cancerous glands (Fig. 1A and B). As previously reported (Darson et al. 1997), the intensity of PSA staining was decreased in prostate cancer in comparison with the BPH tissue sections (Fig. 1C and D). hK2 immunostaining was slightly increased in cancer tissues, again as previously reported (Darson et al. 1999), particularly when compared with the PIN lesion (Fig. 1E and F). No staining was seen in the negative controls (data not shown).
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Establishment of an in vitro kallikrein expression model in stably transfected PC-3 cells
In order to examine the possible functional effects of hK4 in prostate cancer, we established an in vitro expression model in which PC-3 cells were stably transfected with expression constructs for either KLK4, KLK3 or KLK2. PC-3 cells were chosen for this study because they express little or no PSA, hK2 or hK4. RT-PCR analysis confirmed that the clones transfected with KLK4, KLK3 or KLK2 were all positive for the expression of their specific genes (Fig. 2A). The native PC-3 cells and vector-only clones were appropriately negative, although low levels of endogenous KLK4 expression were detected in these cells. Western blotting also showed that hK4, PSA and hK2 protein was overexpressed in the cell lysate and, as typical of these serine proteases, secreted into the medium when compared with the control native or vector-only cells (Fig. 2B). All clones were analysed for changes in proliferation rates, while a selection of clones (asterisked in Fig. 2A) were chosen for the other functional assays.
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After stable transfection, both the hK4- and PSA-expressing PC-3 cells displayed an elongated/irregular morphology (Fig. 2C
). In contrast, cells transfected with the pcDNA3.1:KLK2 or pcDNA3.1:vector-only constructs were similar in structure to the native PC-3 cell line, which had a rounded shape and formed tightly adherent colonies typical of an epithelial, cobblestone appearance.
Subcellular staining of microtubules (green) and actin filaments (red) in each cell line (Fig. 3A) confirmed the small, rounded shape in the majority of native PC-3 cells and those transfected with the empty-vector or pcDNA3.1:KLK2 constructs. The appearance of hK4- and PSA-expressing cell lines was quite distinct from the control cell lines, particularly in the prevalence of small, actin-rich filopodia/microspikes (Fig. 3A; indicated by arrows). More extensive lamellipodia/membrane ruffling (indicated by asterisks) can be observed in these cells than in the native, vector control or hK2-expressing cell lines. Cells with lamellipodia also had classical tubulin staining – dense within the cell body and weak in the lamella (Fig. 3A).
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) confirmed these findings in that the native, vector-only and hK2-expressing PC-3 cell lines revealed distinct focal adhesions with actin and vinculin colocalising to the outer edges of the cell (Fig. 3B; arrowheads). Less concentrated regions of colocalisation were detected in the cells expressing hK4 and PSA. Cell proliferation is decreased in hK4-expressing PC-3 cells, but not altered in PSA- or hK2-expressing cells
In three independent assays, the proliferation of the hK4-expressing PC-3 clones was decreased 2-fold at 96 h (P<0.01) when compared with the parent PC-3 cells (Fig. 4A). No change in the proliferation rate of the PSA- or hK2-expressing clones was observed when compared with control cells at the conclusion of the experimental period.
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None of the prostatic kallikreins expressed in the PC-3 cells significantly induced in vitro invasion through Matrigel compared with the control cell lines (Fig. 4B). In contrast, hK4-transfected PC-3 cells had increased motility at 24 h in comparison with the PC-3 native cells (2.4-fold increase, P<0.01) and vector-only controls (2.9-fold increase, P<0.01) (Fig. 4C). PSA expression in PC-3 cells induced a 2.7-fold increase in motility compared with the vector-only control cell lines (P<0.01) and a 1.7-fold increase in motility compared with the PC-3 native cells at 48 h (P<0.01) (Fig. 4D). Although no change in motility was seen for the hK2-expressing cells compared with the native cells, there was an increase in migration compared with the vector-only controls, but this was not significant. Interestingly, it is noted that the hK4-expressing cells migrated significantly faster (within 24 h) than PC-3 cells expressing PSA and hK2 (measured over 48 h).
Expression of E-cadherin is decreased in response to hK4 and PSA expression in PC-3 cells
These findings suggested that an EMT-like event may have occurred; therefore, the cells were analysed for changes in E-cadherin and vimentin, key markers of epithelial and mesenchymal phenotypes respectively (Savanger 2001). Little or no E-cadherin staining was observed in the hK4- and PSA-expressing cells with an antibody (HECD-1) specific to the E-cadherin ectodomain, while typical patterns of membrane staining at cell–cell contacts were observed in the control cell lines and cells expressing hK2 (Fig. 5A). Identical patterns of expression were found with a second antibody directed to the intracellular domain of E-cadherin (4A2C7; data not shown), indicating that the entire E-cadherin protein was lost from the hK4- and PSA-expressing cell lines. This suggests that the downregulation of E-cadherin may be transcriptional. Real-time PCR analysis for E-cadherin gene expression showed a highly significant 7-fold decrease in PSA-expressing clones and a 10-fold decrease for hK4-expressing clones (Fig. 5B) compared with the PC-3 native cell line (P<0.01) and the vector-only controls (P<0.05). No change was seen in the hK2-expressing cells compared with the control cell lines (P=0.14). As expected for an EMT event, increased staining of vimentin was evident in the cells exhibiting a decrease in E-cadherin (Fig. 5A). These results were confirmed by real-time PCR, demonstrating that vimentin gene expression was elevated in PSA-expressing clones (3.7-fold; P<0.01) and PC-3 cells expressing hK4 (2.3-fold) (Fig. 5B), although this increase for hK4 did not reach statistical significance (P=0.27). Again, there was no significant change in the non-EMT-like, hK2-expressing cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Strikingly, the data presented here also implicate hK4 and PSA, but not hK2, in an epithelial-mesenchymal transition (EMT) process in prostate cancer cells. EMT is a critical event in the progression of most cancers, as epithelial cells lose their characteristic phenotype and dedifferentiate (Thiery 2002). Specifically, migrating epithelial cells often appear spindle-shaped, with loss of cell polarity, reduced cell–cell adhesion and rearrangement of the cytoskeleton (Savanger 2001, Thiery 2002). Observations about the functional effects of these kallikreins are based on the altered morphology and increased motility displayed by the hK4- and PSA-expressing, but not hK2-expressing, PC-3 cells with a concomitant decrease in E-cadherin and increase in vimentin, two well-known EMT markers of epithelial and mesenchymal origin respectively (Savanger 2001).
Although increased levels of serum PSA are associated with metastatic cancer, it is now well documented that PSA gene and protein expression in prostatic cells decreases as tumours become more aggressive and increasingly undifferentiated (Darson et al. 1997, 1999, Tremblay et al. 1997). The reasons for this loss of PSA expression within malignant cells are unclear. Of interest, while the proven metastasis-promoting gene, hepsin, has been associated with overexpression in prostate cancer tissues (Klezovitch et al. 2004, Stephan et al. 2004), its expression has been reported to be lower or even lost in metastatic cancer, suggesting that these genes could exert their effects in the early stages of invasive carcinoma rather than at the metastatic lesion (Vasioukhin 2004). We propose that this may be the case for PSA in prostate cancer progression with a subsequent loss of effects such as the suggested putative antiangiogenic and apoptotic roles (Balbay et al. 1999, Fortier et al. 1999, 2003), which would prevail in the normal functioning of the prostate. Contrary to the immunologic pattern presented by PSA in malignancy, we and several others have observed increased KLK2/hK2 and KLK4/hK4 expression in prostatic tumours (Herrala et al. 2001, Xi et al. 2004). This might suggest that hK4 and hK2 are more highly associated with malignancy than PSA. Furthermore, our migration studies have indicated that while PSA increased motility in PC-3 cells over 48 h, the cells expressing hK4 migrated significantly faster, suggesting that hK4 may be more important than PSA in the motility of cancer cells and ultimately cancer progression.
PC-3 cells were chosen for this study because they express little or no PSA, hK2 or hK4, unlike LNCaP cells. Although PC-3 cells also do not express 
-catenin (Morton et al. 1993), a protein associated with linkage of E-cadherin to the actin cytoskeleton, other molecules, such as vinculin, have been shown to take the place of -catenin, thereby allowing re-establishment of functional, cadherin-mediated cell adhesion (Hazan et al. 1997). Functional consequences of E-cadherin perturbations have also been reported in DU145 prostate cancer cells (Chunthapong et al. 2004), despite the variable expression of -catenin in this cell line. Nonetheless, since E-cadherin was completely lost from the hK4- and PSA-expressing PC-3 cells, post-translational modifications and/or the lack of -catenin are unlikely to be the mechanisms involved in the transcriptional decrease of E-cadherin mRNA and subsequent loss of protein.
As serine proteases, the kallikreins serve post-translationally to cleave specific polypeptide precursors to their bioactive forms, thereby indirectly initiating a range of cellular responses (Borgono & Diamandis 2004, Clements et al. 2004). This study has indicated that such changes may include growth regulation and EMT. A role for prostatic kallikreins in growth regulation has been predicated on the basis of in vitro biochemical analyses of PSA, hK2 and hK4. For instance, all three kallikreins have been shown to degrade insulin-like growth (IGF) factor-binding proteins (IGFBPs) (Clements et al. 2004, Matsumura et al. 2004). This may then increase the bioavailability of the IGFs and therefore their mitogenic action in the prostate microenvironment. From these findings, PSA has been suggested to contribute to cancer cell growth, but, consistent with the findings of Denmeade et al. (2003), this was not demonstrated in our study. Furthermore, hK4 decreased PC-3 cell proliferation in this study, suggesting that it may not have a proliferative role, but instead may activate inhibitors of mitogenic factors or increase apoptosis.
Although it is unclear what level of active protease is secreted by these cells, since chromogenic substrate analysis was inconclusive (data not shown), a number of factors involved in the EMT process are known kallikrein substrates. Increasing levels of transforming growth factor (TGF)ß in patients with metastatic prostate cancer have been correlated with the expression of PSA in serum (Adler et al. 1999). The latent (pro-) form of TGFß is known to be activated by PSA (Killian et al. 1993) and, by prediction, also by hK4 (Matsumura et al. 2004). TGFß has a number of recognised effects on prostate cancer cells, including activation of cytoskeletal modulators, such as the GTPase, RhoA (Bhowmick et al. 2001), and thus rearrangement of the actin cytoskeleton and induction of cell migration. TGFß can also downregulate E-cadherin expression through the transcription factors, Snail and Slug, and promote hypermethylation of the E-cadherin gene, leading to loss of the epithelial phenotype (Savanger 2001, Thiery 2002).
PSA can also activate epidermal growth factor (EGF) (Clements et al. 2004), which can increase cell motility by altering cell polarisation and morphology in fibroblasts (Ware et al. 1998). EGF (Unlu & Leake 2003a), as well as TGFß (Unlu & Leake 2003b), can upregulate urokinase plasminogen activator (uPA) expression and the invasive ability of prostate cancer cells. Recombinant hK4 and hK2 have also been shown to activate the precursor of single-chain uPA (Takayama et al. 2001). Thus, although PC-3 cells intrinsically produce high levels of uPA, this enzyme is unlikely to be the primary mechanism for PSA- or hK4-induced EMT. First, the EMT-like phenotype is not induced by hK2, a known activator of uPA; second, PSA does not activate uPA (Frenette et al. 1997), yet it induces EMT; third, neither PSA nor hK4 induced the PC-3 cells to invade through artificial ECM in vitro above the control cell lines; and, finally, no EMT-like effect was observed in the vector-only controls.
In summary, we have developed in vitro expression models for the prostatic kallikreins, PSA, hK2 and hK4. Characterisation of the phenotype and genotype of these cells clearly indicates, for the first time, that hK4, as well as PSA, has properties that facilitate the progression of prostate cancer cells by modulating regulators of the cytoskeleton and cellular adhesion and migration processes, all hallmarks of the EMT-like processes indicative of aggressive disease.
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Acknowledgements |
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Funding |
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This work was supported by the National Health and Medical Research Council of Australia (grant no. 101202). T Veveris-Lowe and R Collard received Wesley Research Institute and Queensland Cancer Fund postgraduate scholarships respectively. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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Footnotes |
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References |
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