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Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2014-0124 Research Article
Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor Noémie Michel1,a, Nathalie Heuzé-Vourc’h2,a, Elise Lavergne3, Christelle Parent1, Marie-Lise Jourdan4, Amandine Vallet1, Sophie Iochmann2, Orlando Musso3, Pascale Reverdiau2 and Yves Courty1,*
1
INSERM UMR1100, Centre d’Etude des Pathologies Respiratoires, 10 boulevard Tonnellé, F-37032 Tours cedex, France
2
Université François Rabelais, EA6305, Centre d’Etude des Pathologies Respiratoires, 10 boulevard Tonnellé, F-37032 Tours cedex, France
3
INSERM, UMR991, Université de Rennes 1, Rennes, France
4
INSERM UMR 1069, Université François Rabelais, 10 boulevard Tonnellé, F-37032 Tours cedex, France
*Corresponding author e-mail: [email protected]
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Regulation of NSCLC cell growth by KLK6
Abstract The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in nonsmall cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic NSCLC cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.
Keywords: EGFR, kallikrein-related peptidases, lung cancer, PAR.
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Regulation of NSCLC cell growth by KLK6
Introduction Recent advances in cancer research have led to several major breakthroughs, but the overall cancer-related death rate remains unacceptable. Thus, further insight into tumor markers and the development of targeted therapies are urgently needed. The kallikrein-related peptidase (KLK) family contains fifteen serine proteases that are dysregulated in many malignant diseases including prostate, breast, ovarian and lung cancers (Avgeris et al., 2012; HeuzéVourc'h and Courty, 2012). The aberrant expression of KLKs has led to investigations of their potential as cancer biomarkers. Several kallikrein-related peptidases, including KLK3 (PSA, prostate-specific antigen), a well-known marker for prostate cancer, have shown promise as prognostic and predictive cancer biomarkers for: cancer of the prostate, testis, kidney, breast, ovary, lung, colon, pancreas, and brain (reviewed in Schmitt and Magdolen, 2009; Avgeris et al., 2010; Kontos and Scorilas, 2012). Some KLKs have also been involved in many cancerrelated processes, such as cell growth regulation, angiogenesis, invasion, and metastasis (Sotiropoulou et al., 2009Avgeris et al., 2012). Consequently, KLKs are emerging as novel targets for pharmacological intervention (Sotiropoulou and Pampalakis, 2012). KLK6, the gene encoding kallikrein-related peptidase 6, is located within the kallikrein gene cluster on chromosome 19q13. Increased KLK6 mRNA expression has been identified in most epithelial cancers suggesting an oncogenic role for KLK6 (Bayani and Diamandis, 2011). KLK6 has also been found downregulated in breast and brain tumors (Yousef et al., 2004). KLK6 may be involved in malignant cell proliferation, as in lung and colon cancer cells (Heuze-Vourc'h et al., 2009; Kim, J. T. et al., 2011). Other findings support the implication of KLK6 in tumor invasion. KLK6 has the in vitro ability to degrade extracellular matrix proteins including laminin, fibronectin, collagens (Ghosh et al., 2004). In addition, several studies using inhibitors or siRNAs to decrease KLK6 protein expression in model cancer cell lines have revealed a concomitant reduction in the invasive potential of these cells (Shinoda et al., 2007; Henkhaus et al., 2008; Kim, J. T. et al., 2011). Conversely, KLK6 may have a protective function in breast cancer by inhibiting the epithelial-to-mesenchymal transition (Pampalakis et al., 2009). Lastly, the KLK6 gene is inactivated in metastatic breast cancer due to epigenetic events (Pampalakis and Sotiropoulou, 2006). These various findings indicate that KLK6 may exert either cancer-inhibiting or cancer-promoting activities, depending on the tissue type, stage of pathogenesis and tumor microenvironment.
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Regulation of NSCLC cell growth by KLK6 We have previously shown that elevated expression of KLK6 is an indicator of poor prognosis for patients suffering from non-small cell lung cancer (NSCLC). We used an isogenic NSCLC model cell system to demonstrate that ectopic KLK6 expression enhances cell growth by accelerating the cell cycle, between the G1 and S phases (Heuze-Vourc'h et al., 2009). We have now analyzed the way in which KLK6 triggers this response. Our data suggest that KLK6 promotes proliferation and restrains apoptosis in NSCLC cells by stimulating the EGFR signaling pathway and KLK6-dependent activation of the protease-activated receptor 2 (PAR-2).
Results KLK6 peptidase activity and cell growth and survival Ectopic KLK6 expression in cells led to increased growth of isogenic clones of A549 Flp-In (Figure 1A) and HEK293 Flp-In (Figure 1B) cells. Others have reported that proteinases regulate cell growth by mechanisms that are independent of their catalytic activity (Masson et al., 2010). We therefore evaluated the influence of KLK6 proteolytic activity on cell growth in KLK6 isogenic cell systems using a mutant KLK6 in which the serine residue essential for its enzymatic activity was replaced by an alanine residue. The stable expression of this S197A mutant KLK6 hampered KLK6-dependent cell growth (Figure 1C & 1D). Cell growth results to both cell proliferation and cell survival. As we previously reported that KLK6 regulates the growth of NSCLC cells by stimulating cell proliferation (Heuze-Vourc'h et al., 2009), we examined the capacity of this KLK to regulate the survival of A549 cells. Flow cytometry (DNA fragmentation) and ELISA (histone release) analyses revealed that ectopic KLK6 expression significantly reduced the death of staurosporine-stimulated A549 Flp-In cells (Figure 2A, B). This effect was not observed when A549 Flp-In cells were stably transduced with a vector encoding the S197A mutant (Figure 2C). These data suggest that the KLK6mediated growth of NSCLC cells is due to both proliferation and resistance to apoptosis, and is dependent on the enzymatic activity of KLK6.
KLK6 and the nuclear translocation of β-catenin Enhanced cell proliferation was reported to be associated with the down-regulation of E-cadherin and the accumulation of β-catenin in the nuclei of mouse keratinocyte cells stably
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Regulation of NSCLC cell growth by KLK6 transfected with Klk6 (Klucky et al., 2007). We used immunofluorescence analysis of controls and stable A549-KLK6 cells with an anti-β-catenin antibody to determine whether ectopic KLK6 expression modified distribution of β-catenin in NSCLC cells. β-catenin was mainly located at the cell membrane in control cells (Figure 3A) while its distribution was diffuse in A549-KLK6 cells (Figure 3B). This difference in β-catenin protein distribution was not due to altered transcription because quantitative RT-PCR revealed comparable amounts of transcripts in the control and A549-KLK6 cells (Figure 3C). Free β-catenin may be rapidly degraded, kept in the cytoplasm or translocated to the nucleus where it becomes associated with TCF/Lef family transcription factors and drives the transcription of TCF/LEF-1 target genes (Valenta et al., 2012). We therefore transfected cells with a TCF-dependent luciferase reporter plasmid (TCFwt-luci) to determine whether the ectopic KLK6 was associated with activation of β-catenin/TCF-dependent transcription. The reporter gene was twice as active in transfected A549-KLK6 cells as in control cells (Figure 3D). In contrast, no difference was measured for a luciferase reporter plasmid (TCFmut-luci) with inactive TCF-binding sites. Activation of the canonical Wnt pathway or altered E-cadherin-β-catenin association often results in the accumulation of β-catenin. This, in turn, leads to enhanced TCF/LEF-1-driven transcription. In mouse keratinocytes, β-catenin translocation induced by Klk6 follows shedding of an E-cadherin ectodomain (Klucky et al., 2007). Our Western blot analyses showed similar amounts of full-length (Figure 3E) and soluble E-cadherin (Figure 3F) in whole-cell extracts and conditioned medium from both control and A549-KLK6 cells. This suggests that KLK6 does not modify the synthesis of E-cadherin or the shedding of its ectodomain in NSCLC cells and that the translocation of β-catenin to the nucleus results from a different mechanism. Recent studies have shown that EGFR activation can also lead to the translocation of β-catenin to the nucleus in certain carcinomas (Lee et al., 2010; Ma et al., 2013). We therefore examined this pathway in NSCLC cells.
KLK6 triggers the EGFR pathway through activation of PAR-2 We treated our clones with Cetuximab to investigate whether EGFR could regulate KLK6dependent cell growth. This chimeric mouse-human antibody targets the extracellular domain of EGFR and prevents the binding of activating ligands to the EGFR. Treatment of A549 FlpIn cells with Cetuximab blocked KLK6-dependent cell growth (Figure 4A). Because EGFR signaling acts mainly through the Raf/Mek/ERK and PI3K/AKT pathways, we examined these pathways in cells that had been transiently exposed to KLK6. Addition of recombinant
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Regulation of NSCLC cell growth by KLK6 KLK6 (10 µg/ml) in the medium activated the phosphorylation of ERK in control cells (Figure 4B). This activation was inhibited by Cetuximab. Similarly, the transient expression of KLK6 stimulated the phosphorylation of Akt at Thr308 in control cells, and this activation was suppressed by cetuximab (Figure 4C). Similar results were obtained when Cetuximab was replaced by the EGFR tyrosine kinase inhibitor AG1478 (Figure 4D). As PAR2 has been described as a substrate of KLK6 (Angelo et al., 2006; Oikonomopoulou et al., 2006), we examined the influence of activating PAR2 on the ligand-dependent activation of the EGFR pathway in A549 Flp-In cells. Activation of PAR2 with an agonist peptide stimulated the growth of these NSCLC cells (Figure 5A) by a mechanism involving the binding of a ligand to EGFR. Indeed, the PAR2-dependent phosphorylation of ERK was abolished by treatment with Cetuximab (Figure 5B). We used A549 Flp-In transfected with short-interfering RNAs against PAR2 to determine whether PAR2 is involved in the response to KLK6. A nonsilencing sequence was used as a negative control. The expression of both PAR2 mRNA and protein was reduced by 90% in cells treated with PAR2-siRNA compared to that of cells treated with control-siRNA (Figure 5C). Transfection of the cells with PAR– siRNA abolished the PAR2 agonist peptide-dependent phosphorylation of ERK (Figure 5 D). Similarly, the wt.KLK6-dependent phosphorylation of ERK and Akt (Figure 6 A, B) was inhibited in the presence of a siRNA against PAR2.
Discussion KLK6 expression has been associated with a poor prognosis for patients with ovarian (Kountourakis et al., 2008; White et al., 2009; Seiz et al., 2012), gastric (Nagahara et al., 2005; Kim, J. J. et al., 2012), colon (Kim, J. T. et al., 2011) or lung cancers (Heuze-Vourc'h et al., 2009) and with glioblastoma multiforme (Drucker et al., 2013) suggesting that this protease contributes to tumor progression. The expression of KLK6 has also been connected with the increased growth and invasion of several malignant cells (Nagahara et al., 2005; Henkhaus et al., 2008; Kim, J. T. et al., 2011; Krenzer et al., 2011; Kim, J. J. et al., 2012). Our studies showed that the ectopic production of KLK6 accelerates the cell cycle, between the G1 and S phases, of NSCLC cells (Heuze-Vourc'h et al., 2009) and decreases apoptosis (described in this study). The ability of KLK6 to act on these two processes is supported by previous observations. Silencing the KLK6 gene effectively suppressed the rate at which a gastric cell line proliferated and decreased the cell population in the S phase (Nagahara et al.,
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Regulation of NSCLC cell growth by KLK6 2005). KLK6 has also been shown to enhance the survival of immune and glioblastoma cells (Scarisbrick et al., 2011; Drucker et al., 2013). Lastly, Kupferman found that KLK6 overproduction was associated with the anoikis-resistant phenotype of oral cancer cell lines (Kupferman et al., 2006). Our data indicate that a catalytically inactive mutant did not modify cell processes and signaling in the way that wild-type KLK6 did. This suggests that the active form is involved in the above processes. We believe that the zymogen secreted by the transfected cells was processed in the cell-culture medium. KLK6 was initially reported to be self-activated (Bayes et al., 2004) but subsequent studies revealed that KLK6 was essentially unable to activate proKLK6, suggesting another protease activates KLK6 (Blaber et al., 2007). The best activator of pro-KLK6 identified to date is plasmin (Yoon et al., 2008). It is conceivable that a cascade involving plasmin has generated active KLK6 in our culture conditions because the plasmin system is functional when A549 cells are cultured in the presence of serum (Van Leer et al., 2005). Both chronic and transient exposures to KLK6 modify several signaling pathways including the ERK and Akt pathways. As phosphorylation of these kinases is inhibited by silencing PAR-2, we consider that KLK6 initially signals through this G protein-coupled receptor. There is now good evidence indicating that KLKs are activators of PARs (reviewed in Oikonomopoulou et al., 2010; Caliendo et al., 2012). In particular, KLK4 has been associated with prostate cancer progression, and this effect has been linked to PAR1 and 2 (Mize et al., 2008; Ramsay et al., 2008). KLK4 signaling via PAR1 and KLK14 signaling via PAR2 have also been involved in colon tumorigenesis (Gratio et al., 2010; Gratio et al., 2011; Chung et al., 2013). Several studies have demonstrated that KLK6 can signal through PARs, mainly in the central nervous system. A KLK6-evoked intracellular Ca2+ flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes (Vandell et al., 2008). The KLK6mediated activation of PAR1 is also involved in promoting lymphocyte survival and the survival and stellation of astrocytes (Scarisbrick et al., 2011; Scarisbrick et al., 2012; Drucker et al., 2013). Finally, KLK6 plays a critical role in oligodendrogliopathy and neurodegeneration through PAR1 and PAR2 signaling (Burda et al., 2013; Yoon et al., 2013). PAR-2 stimulation has several effects on A549 cells, including the release of IL-6, IL-8 and prostaglandin E2 (PGE2), induction of COX-2 and reduction of apoptosis (Moriyuki et al., 2008; Moriyuki et al., 2009; Huang et al., 2013). PGE2 formation has been attributed to activation of the ERK/ PI3K / Akt / NF-kappaB pathway while the PAR2-triggered increase 7 / 24
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Regulation of NSCLC cell growth by KLK6 in COX-2 was described as being partly dependent on the ERK1/2-mediated activation of the beta-catenin/TCF-4 and CREB pathways (Moriyuki et al., 2008; Moriyuki et al., 2009). The signaling pathways (ERK, Akt, β-catenin/TCF) that respond to KLK6 therefore agree with those previously described in A549 cells following activation of PAR-2. Thus, these data support our belief that KLK6 drives some A549 cell functions via PAR2. As the gene c-myc is a target of TCF/LEF-1, the increase in β-catenin/TCF-dependent transcription we have observed correlates with our published data showing that KLK6 triggers c-myc induction in the same A549-FlpIn wt.KLK6 clone (Heuze-Vourc'h et al., 2009). The PAR2 agonist peptide appeared less efficient than KLK6 for promoting cell growth. This suggests that either KLK6 directly or indirectly targets other factors involved in these pathways or the canonical agonist peptide does not mimic all actions of KLK6 on PAR2. This last assumption is supported by several recent studies showing that the PARs are capable of ‘functional selectivity’ or ‘biased’ signaling (reviewed in Hollenberg et al., 2014). Darmoul and collaborators were the first to report that PAR2 could transactivate EGFR (Darmoul et al., 2004). Stimulation of PAR2 in colon cancer cells with trypsin resulted in the MMP-mediated release of an EGF family ligand (TGF- ), which in turn activated EGFR. More recent studies have reported the KLK1-triggered PAR1-dependent transactivation of EGFR in keratinocytes and neurons (Gao et al., 2009; Gao et al., 2010; Lu et al., 2014). Our results suggest that KLK6 promotes the PAR-2-dependent transactivation of EGFR. Both Cetuximab and AG1478, two inhibitors of the EGFR pathways, blocked the KLK6dependent phosphorylation of ERK and Akt. The inhibition by Cetuximab indicates that transactivation of the EGFR pathway is probably ligand-dependent. Further studies are required to identify the molecules involved in transmission of the signal between PAR-2 and EGFR. This process usually implicates members of the disintegrin and metalloproteinase (ADAM) family that perform the shedding of the ectodomain of membrane-bound EGFR ligands such as TGF-α (transforming growth factor-α), HB-EGF (heparin-binding EGF-like growth factor) and amphiregulin (Kataoka, 2009). A second mechanism for transactivation of the EGFR by PAR2 signaling has been described in some circumstances {Caruso et al., 2006; van der Merwe et al., 2008}. Stimulation of PAR2 may cause activation of Src which in turn leads, via an intracellular route, to the tyrosine phosphorylation and subsequent activation of the EGFR {Caruso, 2006 #2362; van der Merwe, 2008 #2361}. Whether this alternative pathway is involved in the KLK6-dependent transactivation of the EGFR remains to be determined.
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Regulation of NSCLC cell growth by KLK6 In summary, our results suggest that KLK6 promotes the proliferation of NSCLC cells and restricts their apoptosis via an activation cascade initiated by PAR2 and involving the liganddependent transactivation of EGFR.
Materials and methods Materials HEK-293 Flp-In cells, cell culture media, antibiotics, Lipofectamine, cloning plasmids, SuperScript VILO cDNA kit and Alexa Fluor 488 were all from Invitrogen (Fisher Bioblock Scientific, Illkirch, France). Parental A549 cells were from the American Type Culture Collection. Fetal calf serum (FCS) was from Lonza (Verviers, Belgium). TOP-flash plasmid DNA and negative control FOP-flash plasmid DNA were purchased from Addgene (Cambridge, USA). SYBR® Premix Ex TaqTM kit and siRNA PAR2 were from Takara (Ozyme, St-Quentin en Yvelines, France) and Applied Biosystems (Courtaboeuf, France), respectively. CellTiter 96® Aqueous One Solution Cell Proliferation Assay and DualLuciferase Assay System were from Promega France (Charbonnieres les Bains, France). Cell Death Detection ELISA kit was from Roche Molecular Biochemicals (Meylan, France). AntissDNA/APOSTAIN F7-26 monoclonal antibody was from Eurobio-Abcys (Courtaboeuf, France). FITC-conjugated anti-mouse IgM antibody was from Sigma-Aldrich (Saint-Quentin Fallavier, France). Anti-β-catenin, Anti- soluble E-cadherin, phospho-Erk1/2 and total Erk1/ 2 antibodies were from Santa-Cruz Biotechnology (CliniSciences, Nanterre, France). Antiphospho-Akt (Thr 308; Ser 473) and anti-Akt were from Cell Signalling Technology (Ozyme, France). Cetuximab/Erbitux was from Merck Serono (Lyon, France). Other reagents were from Sigma-Aldrich. Recombinant KLK6 was produced, purified and activated as previously described (Oikonomopoulou et al., 2008).
Culture conditions and cell viability assay A549 Flp-In cells and the A549 Flp-In clone expressing wt.KLK6 were previously described (Heuze-Vourc'h et al., 2009). The A549 Flp-In cells were grown in RPMI-1640 Glutamax and the HEK293 Flp-In cells were grown in D-MEM. The media were supplemented with 10% FCS, 2mM L-glutamine, 100U/ml penicillin and either 100 µg/ml zeocin or 100 µg/ml hygromycin. The cells used in the viability assay were seeded in 96-well plate and maintained
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Regulation of NSCLC cell growth by KLK6 at 37 °C for an appropriate time. Medium was then removed and cells were incubated for 2 h at 37 °C in 100µl culture medium and 20µl CellTiter 96® Aqueous One Solution Cell Proliferation reagent containing MTS. The absorbance at 490 nm was determined with a microplate reader.
Apoptosis analysis Cells were seeded in 96-well plate (ELISA) at 5×103 cells per well or in six-well plates (APOSTAIN) at 5×105 cells per well and incubated for 16 hours. The medium was then collected and the cells incubated in RPMI, 2% FCS with 0.2 µM staurosporine for 24h. Apoptosis was measured with the Cell Death Detection ELISA kit in accordance with the manufacturer's instructions. The cells used in the APOSTAIN analysis were fixed with icecold methanol and collected by centrifugation. They were then suspended in 250 μl formamide for 5 min at room temperature and incubated at 75°C for 10 min. Then, 2 ml of PBS containing 1% of nonfat dried milk were added and cells were incubated for 15 min at room temperature, collected by centrifugation and incubated for 15 min in 75 μl PBS containing 5% FCS and anti-ssDNA F7-26 monoclonal antibody. These cells recovered by centrifugation and incubated for 15 min in 100 μl PBS containing FITC-conjugated antimouse IgM (20 μg/ml) and 1% nonfat dried milk. Cells were rinsed, centrifuged, and resuspended in 200 μl of PBS containing 1 μg/mL of 7-AAD. Negative controls were treated with IgM instead of the specific primary antibody. All cells were then analyzed by flow cytometry.
Immunofluorescence analysis A549 cells were grown to 90% confluence in LAB-TEK chamber slides, fixed with ice-cold methanol for 10 minutes and wash repeatedly with PBS (pH 7.2). These fixed cells were incubated for 30 min with blocking buffer (PBS plus 1% bovine serum albumin (BSA), then for 2 h with anti-β-catenin antibody diluted to 4µg/ml in incubation buffer (1% BSA in PBS) and washed thoroughly with PBS. Finally, they were incubated for 1 h with Alexa Fluor 488 diluted in incubation buffer, washed
with PBS, and mounted with mounting medium
(IF fluoromount G) on glass slides. Images were acquired by confocal microscopy.
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Regulation of NSCLC cell growth by KLK6
Isolation of total RNA and real-time PCR analysis Total RNA was extracted using Total ARN Isolation Nucleospin RNA II kits according to the manufacturer‘s instructions (Macherey-Nagel, Hoerdt, France). The eluted RNA was quantified by spectrophotometry and 5 μg aliquots were reverse-transcribed to obtain the corresponding cDNAs with SuperScript VILO cDNA kits. Real-time quantitative PCR was performed with SYBR® Premix Ex TaqTM kits using the Roche LightCycler 480 instrument. The primer sets were: for β-catenin 5′-CAC AAG CAG AGT CCA AAG ACA G-3′ and 5′GAT TCC TGA GAG TCC AAA GAC AG-3′, for β-actin 5′-GAC CAT TGG CAA TGA GCG GTT C-3′ and 5′-AGG TCT TTG CGG ATG TCC ACG T-3′, and for PAR2 5’-CTC CTC TCT GTC ATC TGG TTC C-3’ and 5’-TGC ACA CTG AGG CAG GTC ATG A-3’.
Luciferase reporter assay A549 Flp-In and A549-KLK6 cells were seeded in 24-well plates and incubated for 16h as above. They were then transfected with the TOP-flash plasmid DNA (TCFwt) or the negative control FOP-flash plasmid DNA (TCFmut) together with Renilla luciferase-coding plasmids by incubation for 4 h with Lipofectamine Plus TM in serum and antibiotic-free OptiMEM. The medium was changed to RPMI plus antibiotics and 10% FCS and the cells grown for a further 16 h. The Firefly and Renilla luciferase activities in these cells were measured using the Dual-Luciferase Assay System according to the manufacturer’s instructions. Firefly luciferase was normalized to Renilla luciferase activity.
Western blot analysis Whole cell extracts were prepared with lysis buffer A (200 mM Tris pH8, 137 mM NaCl, 1% NP40, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, proteinase inhibitor cocktail). Equal amounts of protein (cell lysates or supernatants) were loaded onto SDSpolyacrylamide gels, separated, and the resulting proteins transferred to polyvinylidene fluoride membranes, whose free sites had been blocked by incubation for 1 h at room temperature with TBS Tween-20 buffer containing 5% dried milk (soluble E-cadherin and phospho-Erk1/2) or 1% BSA (total Erk1/2). The membranes were then incubated with primary antibodies [anti-soluble E-cadherin (1:1000), anti-phospho-Erk1/2 (1:500), anti-total Erk1/2 (1:1000)] overnight at 4°C. The blots were incubated with horseradish peroxidase (HRP) labeled anti-mouse immunoglobulin G or anti-rabbit immunoglobulin G (1:5000) at
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Regulation of NSCLC cell growth by KLK6 room temperature for 2 hrs in TBS Tween-20 containing 5% dried milk. Proteins were revealed by enhanced chemiluminescence.
siRNA transfection A549 cells (0.3×106 cells / well in 6-well plates) were incubated overnight and then transfected by incubation with 12.5 pmol of a small interfering RNA (siRNA) that targeted PAR2 transcripts in OptiMEM plus Lipofectamine for 4 h. Control transfections were performed with an nonsilencing control siRNA. The transfected cells were washed once with PBS and then incubated for 24 h in RPMI plus 10% FCS. The medium was changed and the cells were treated with cetuximab and/or PAR2 agonist.
Statistics All statistical analyses were performed with GraphPad Prism 5.00 (GraphPad Software Inc., San Diego, CA, USA). Results are given as means ± SEM of at least 3 independent experiments.
Acknowledgements The English text was edited by Dr. Owen Parkes. Financial support was provided by the Ligue Contre le Cancer (Comités d’Indre et d’Eure et Loire) and Région Centre (grant KalliCap). Noémie Michel was the recipient of a Fellowship from the Région Centre.
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Regulation of NSCLC cell growth by KLK6
References Angelo, P. F., Lima, A. R., Alves, F. M., Blaber, S. I., Scarisbrick, I. A., Blaber, M., Juliano, L., and Juliano, M. A. (2006). Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects. J. Biol. Chem. 281, 3116-3126. Avgeris, M., Mavridis, K., and Scorilas, A. (2010). Kallikrein-related peptidase genes as promising biomarkers for prognosis and monitoring of human malignancies. Biol. Chem. 391, 505-511. Avgeris, M., Mavridis, K., and Scorilas, A. (2012). Kallikrein-related peptidases in prostate, breast, and ovarian cancers: from pathobiology to clinical relevance. Biol. Chem. 393, 301317. Bayani, J., and Diamandis, E. P. (2011). The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6). Clin. Chem. Lab. Med. 50, 211-233. Bayes, A., Tsetsenis, T., Ventura, S., Vendrell, J., Aviles, F. X., and Sotiropoulou, G. (2004). Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation. Biol. Chem. 385, 517-524. Blaber, S. I., Yoon, H., Scarisbrick, I. A., Juliano, M. A., and Blaber, M. (2007). The autolytic regulation of human kallikrein-related peptidase 6. Biochemistry 46, 5209-5217. Burda, J. E., Radulovic, M., Yoon, H., and Scarisbrick, I. A. (2013). Critical role for PAR1 in kallikrein 6-mediated oligodendrogliopathy. Glia 61, 1456-1470. Caliendo, G., Santagada, V., Perissutti, E., Severino, B., Fiorino, F., Frecentese, F., and Juliano, L. (2012). Kallikrein Protease Activated Receptor (PAR) Axis: An Attractive Target for Drug Development. J. Med. Chem. 55, 6669-6686. Caruso, R., Pallone, F., Fina, D., Gioia, V., Peluso, I., Caprioli, F., Stolfi, C., Perfetti, A., Spagnoli, L. G., Palmieri, G., et al. (2006). Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation. Am J Pathol. 169, 268-278. Chung, H., Ramachandran, R., Hollenberg, M. D., and Muruve, D. A. (2013). Proteinaseactivated Receptor-2 Transactivation of Epidermal Growth Factor Receptor and Transforming Growth Factor-beta Receptor Signaling Pathways Contributes to Renal Fibrosis. J. Biol. Chem. 288, 37319-37331. Darmoul, D., Gratio, V., Devaud, H., and Laburthe, M. (2004). Protease-activated receptor 2 in colon cancer: trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation. J. Biol. Chem. 279, 20927-20934 Drucker, K. L., Paulsen, A. R., Giannini, C., Decker, P. A., Blaber, S. I., Blaber, M., Uhm, J. H., O'Neill, B. P., Jenkins, R. B., and Scarisbrick, I. A. (2013). Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma. Neurol. Oncol. 15, 305-318. Gao, L., Chao, L., and Chao, J. (2009). A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor. Exp. Cell Res. 316, 376-389. Gao, L., Chao, L., and Chao, J. (2010). A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor. Exp. Cell Res. 316, 376-389.
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Regulation of NSCLC cell growth by KLK6 Ghosh, M. C., Grass, L., Soosaipillai, A., Sotiropoulou, G., and Diamandis, E. P. (2004). Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol. 25, 193-199. Gratio, V., Beaufort, N., Seiz, L., Maier, J., Virca, G. D., Debela, M., Grebenchtchikov, N., Magdolen, V., and Darmoul, D. (2010). Kallikrein-related peptidase 4: a new activator of the aberrantly expressed protease-activated receptor 1 in colon cancer cells. Am. J. Pathol. 176, 1452-1461. Gratio, V., Loriot, C., Duke Virca, G., Oikonomopoulou, K., Walker, F., Diamandis, E. P., Hollenberg, M. D., and Darmoul, D. (2011). Kallikrein-Related Peptidase 14 Acts on Proteinase-Activated Receptor 2 to Induce Signaling Pathway in Colon Cancer Cells. Am. J. Pathol. 179, 2625-2636. Henkhaus, R. S., Gerner, E. W., and Ignatenko, N. A. (2008). Kallikrein 6 is a mediator of KRAS-dependent migration of colon carcinoma cells. Biol. Chem. 389, 757-764. Heuzé-Vourc'h, N., and Courty, Y. (2012). Pathophysiology of Kallikrein-related Peptidase in Lung Cancer. In: Kallikrein-Related Peptidases, V. Magdolen, C.P. Sommerhoff, H. Fritz, and M. Schmitt, eds. (De Gruyter), pp. 3-26. Heuze-Vourc'h, N., Planque, C., Guyetant, S., Coco, C., Brillet, B., Blechet, C., Parent, C., Briollais, L., Reverdiau, P., Jourdan, M. L., and Courty, Y. (2009). High Kallikrein-Related Peptidase 6 in Non-Small Cell Lung Cancer Cells: an indicator of tumor proliferation and poor prognosis. J. Cell. Mol. Med. 13, 4014-4022. Huang, S. H., Li, Y., Chen, H. G., Rong, J., and Ye, S. (2013). Activation of proteinaseactivated receptor 2 prevents apoptosis of lung cancer cells. Cancer Invest. 31, 578-581. Kataoka, H. (2009). EGFR ligands and their signaling scissors, ADAMs, as new molecular targets for anticancer treatments. J. Dermatol. Sci. 56, 148-153. Hollenberg, M. D., Mihara, K., Polley, D., Suen, J. Y., Han, A., Fairlie, D. P., and Ramachandran, R. (2014). Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease. Br. J. Pharmacol. 171, 1180-1194. Kim, J. J., Kim, J. T., Yoon, H. R., Kang, M. A., Kim, J. H., Lee, Y. H., Kim, J. W., Lee, S. J., Song, E. Y., Myung, P. K., and Lee, H. G. (2012). Upregulation and secretion of kallikreinrelated peptidase 6 (KLK6) in gastric cancer. Tumour Biol. 33, 731-738. Kim, J. T., Song, E. Y., Chung, K. S., Kang, M. A., Kim, J. W., Kim, S. J., Yeom, Y. I., Kim, J. H., Kim, K. H., and Lee, H. G. (2011). Up-regulation and clinical significance of serine protease kallikrein 6 in colon cancer. Cancer 117, 2608-2619 Klucky, B., Mueller, R., Vogt, I., Teurich, S., Hartenstein, B., Breuhahn, K., Flechtenmacher, C., Angel, P., and Hess, J. (2007). Kallikrein 6 induces E-cadherin shedding and promotes cell proliferation, migration, and invasion. Cancer Res. 67, 8198-8206. Kontos, C. K., and Scorilas, A. (2012). Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers. Clin. Chem. Lab. Med. 50, 1877-1891. Kountourakis, P., Psyrri, A., Scorilas, A., Camp, R., Markakis, S., Kowalski, D., Diamandis, E. P., and Dimopoulos, M. A. (2008). Prognostic value of kallikrein-related peptidase 6 protein expression levels in advanced ovarian cancer evaluated by automated quantitative analysis (AQUA). Cancer Sci. 99, 2224-2229.
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Regulation of NSCLC cell growth by KLK6 Krenzer, S., Peterziel, H., Mauch, C., Blaber, S. I., Blaber, M., Angel, P., and Hess, J. (2011). Expression and function of the kallikrein-related peptidase 6 in the human melanoma microenvironment. J. Invest. Dermatol. 131, 2281-2288. Kupferman, M. E., Patel, V., Sriuranpong, V., Amornphimoltham, P., Jasser, S. A., Mandal, M., Zhou, G., Wang, J., Coombes, K., Multani, A., et al. (2006). Molecular analysis of anoikis resistance in oral cavity squamous cell carcinoma. Oral Oncol. 43, 440-454. Lee, C. H., Hung, H. W., Hung, P. H., and Shieh, Y. S. (2010). Epidermal growth factor receptor regulates beta-catenin location, stability, and transcriptional activity in oral cancer. Mol. Cancer 9, 64. Lu, Z., Cui, M., Zhao, H., Wang, T., Shen, Y., and Dong, Q. (2014). Tissue kallikrein mediates neurite outgrowth through epidermal growth factor receptor and flotillin-2 pathway in vitro. Cell. Signal. 26, 220-232. Ma, L., Zhang, G., Miao, X. B., Deng, X. B., Wu, Y., Liu, Y., Jin, Z. R., Li, X. Q., Liu, Q. Z., Sun, D. X., et al. (2013). Cancer stem-like cell properties are regulated by EGFR/AKT/βcatenin signaling and preferentially inhibited by gefitinib in nasopharyngeal carcinoma. FEBS J. 280, 2027-2041. Masson, O., Bach, A. S., Derocq, D., Prebois, C., Laurent-Matha, V., Pattingre, S., and Liaudet-Coopman, E. (2010). Pathophysiological functions of cathepsin D: targeting its catalytic activity versus its protein binding activity? Biochimie 92, 1635-1643. Mize, G. J., Wang, W., and Takayama, T. K. (2008). Prostate-specific kallikreins-2 and -4 enhance the proliferation of DU-145 prostate cancer cells through protease-activated receptors-1 and -2. Mol. Cancer Res. 6, 1043-1051. Moriyuki, K., Nagataki, M., Sekiguchi, F., Nishikawa, H., and Kawabata, A. (2008). Signal transduction for formation/release of interleukin-8 caused by a PAR2-activating peptide in human lung epithelial cells. Regul. Pept. 145, 42-48. Moriyuki, K., Sekiguchi, F., Matsubara, K., Nishikawa, H., and Kawabata, A. (2009). Proteinase-activated receptor-2-triggered prostaglandin E(2) release, but not cyclooxygenase-2 upregulation, requires activation of the phosphatidylinositol 3-kinase/Akt / nuclear factor-κB pathway in human alveolar epithelial cells. J. Pharmacol. Sci. 111, 269275. Nagahara, H., Mimori, K., Utsunomiya, T., Barnard, G. F., Ohira, M., Hirakawa, K., and Mori, M. (2005). Clinicopathologic and biological significance of kallikrein 6 overexpression in human gastric cancer. Clin. Cancer Res. 11, 6800-6806. Oikonomopoulou, K., Diamandis, E. P., and Hollenberg, M. D. (2010). Kallikrein-related peptidases: proteolysis and signaling in cancer, the new frontier. Biol. Chem. 391, 299-310. Oikonomopoulou, K., Hansen, K. K., Baruch, A., Hollenberg, M. D., and Diamandis, E. P. (2008). Immunofluorometric activity-based probe analysis of active KLK6 in biological fluids. Biol. Chem. 389, 747-756. Oikonomopoulou, K., Hansen, K. K., Saifeddine, M., Tea, I., Blaber, M., Blaber, S. I., Scarisbrick, I., Andrade-Gordon, P., Cottrell, G. S., Bunnett, N. W., et al. (2006). Proteinase-activated receptors, targets for kallikrein signaling. J. Biol. Chem. 281, 3209532112. Pampalakis, G., Prosnikli, E., Agalioti, T., Vlahou, A., Zoumpourlis, V., and Sotiropoulou, G. (2009). A tumor-protective role for human kallikrein-related peptidase 6 in breast cancer mediated by inhibition of epithelial-to-mesenchymal transition. Cancer Res. 69, 3779-3787. 15 / 24
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Regulation of NSCLC cell growth by KLK6 Pampalakis, G., and Sotiropoulou, G. (2006). Multiple mechanisms underlie the aberrant expression of the human kallikrein 6 gene in breast cancer. Biol. Chem. 387, 773-782. Ramsay, A. J., Dong, Y., Hunt, M. L., Linn, M., Samaratunga, H., Clements, J. A., and Hooper, J. D. (2008). Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression. J. Biol. Chem. 283, 12293-12304. Scarisbrick, I. A., Epstein, B., Cloud, B. A., Yoon, H., Wu, J., Renner, D. N., Blaber, S. I., Blaber, M., Vandell, A. G., and Bryson, A. L. (2011). Functional role of kallikrein 6 in regulating immune cell survival. PLoS One 6, e18376. Scarisbrick, I. A., Radulovic, M., Burda, J. E., Larson, N., Blaber, S. I., Giannini, C., Blaber, M., and Vandell, A. G. (2012). Kallikrein 6 is a novel molecular trigger of reactive astrogliosis. Biol. Chem. 393, 355-367. Schmitt, M., and Magdolen, V. (2009). Using kallikrein-related peptidases (KLK) as novel cancer biomarkers. Thromb. Haemost. 101, 222-224. Seiz, L., Dorn, J., Kotzsch, M., Walch, A., Grebenchtchikov, N. I., Gkazepis, A., Schmalfeldt, B., Kiechle, M., Bayani, J., Diamandis, E. P., et al. (2012). Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients. Biol. Chem. 393, 391-401. Shinoda, Y., Kozaki, K., Imoto, I., Obara, W., Tsuda, H., Mizutani, Y., Shuin, T., Fujioka, T., Miki, T., and Inazawa, J. (2007). Association of KLK5 overexpression with invasiveness of urinary bladder carcinoma cells. Cancer Sci. 98, 1078-1086. Sotiropoulou, G., and Pampalakis, G. (2012). Targeting the kallikrein-related peptidases for drug development. Trends Pharmacol. Sci. 33, 623-634. Sotiropoulou, G., Pampalakis, G., and Diamandis, E. P. (2009). Functional roles of human kallikrein-related peptidases. J. Biol. Chem. 284, 32989-32994. Valenta, T., Hausmann, G., and Basler, K. (2012). The many faces and functions of β-catenin. EMBO J. 31, 2714-2736. Van der Merwe, J. Q., Hollenberg, M. D., and MacNaughton, W. K. (2008). EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 294, G441451. Van Leer, C., Stutz, M., Haeberli, A., and Geiser, T. (2005). Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro. Thromb. Haemost. 94, 1257-1264. Vandell, A. G., Larson, N., Laxmikanthan, G., Panos, M., Blaber, S. I., Blaber, M., and Scarisbrick, I. A. (2008). Protease-activated receptor dependent and independent signaling by kallikreins 1 and 6 in CNS neuron and astroglial cell lines. J. Neurochem. 107, 855-870 White, N. M., Mathews, M., Yousef, G. M., Prizada, A., Popadiuk, C., and Dore, J. J. (2009). KLK6 and KLK13 predict tumor recurrence in epithelial ovarian carcinoma. Br. J. Cancer 101, 1107-1113. Yoon, H., Blaber, S. I., Evans, D. M., Trim, J., Juliano, M. A., Scarisbrick, I. A., and Blaber, M. (2008). Activation profiles of human kallikrein-related peptidases by proteases of the thrombostasis axis. Protein Sci. 17, 1998-2007.
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Regulation of NSCLC cell growth by KLK6 Yoon, H., Radulovic, M., Wu, J., Blaber, S. I., Blaber, M., Fehlings, M. G., and Scarisbrick, I. A. (2013). Kallikrein 6 signals through PAR1 and PAR2 to promote neuron injury and exacerbate glutamate neurotoxicity. J. Neurochem. 127, 283-298. Yousef, G. M., Borgono, C. A., White, N. M., Robb, J. D., Michael, I. P., Oikonomopoulou, K., Khan, S., and Diamandis, E. P. (2004). In silico analysis of the human kallikrein gene 6. Tumour Biol. 25, 282-289.
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Regulation of NSCLC cell growth by KLK6
Figures
Figure 1
Active KLK6 promotes cell growth, while the mutant KLK6 form does not.
(A and B) The growth of parental Flp-In cells (dashed lines) and isogenic clones stably producing wild-type KLK6 (full lines) was evaluated by counting cells at the indicated times; (A) A549 Flp-In cells, (B) HEK293 Flp-In cells. (C) A549 and (D) HEK-293 cells stably producing wild-type KLK6 (wt.KLK6) or the S197A mutant (mut.KLK6) were seeded at the same density and cell growth was measured after 48h using an MTS proliferation assay. Values are means ± SEM. Krustal-Wallis test, *P
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Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2014-0124 Research Article
Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor Noémie Michel1,a, Nathalie Heuzé-Vourc’h2,a, Elise Lavergne3, Christelle Parent1, Marie-Lise Jourdan4, Amandine Vallet1, Sophie Iochmann2, Orlando Musso3, Pascale Reverdiau2 and Yves Courty1,*
1
INSERM UMR1100, Centre d’Etude des Pathologies Respiratoires, 10 boulevard Tonnellé, F-37032 Tours cedex, France
2
Université François Rabelais, EA6305, Centre d’Etude des Pathologies Respiratoires, 10 boulevard Tonnellé, F-37032 Tours cedex, France
3
INSERM, UMR991, Université de Rennes 1, Rennes, France
4
INSERM UMR 1069, Université François Rabelais, 10 boulevard Tonnellé, F-37032 Tours cedex, France
*Corresponding author e-mail: [email protected]
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Regulation of NSCLC cell growth by KLK6
Abstract The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in nonsmall cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic NSCLC cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.
Keywords: EGFR, kallikrein-related peptidases, lung cancer, PAR.
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Regulation of NSCLC cell growth by KLK6
Introduction Recent advances in cancer research have led to several major breakthroughs, but the overall cancer-related death rate remains unacceptable. Thus, further insight into tumor markers and the development of targeted therapies are urgently needed. The kallikrein-related peptidase (KLK) family contains fifteen serine proteases that are dysregulated in many malignant diseases including prostate, breast, ovarian and lung cancers (Avgeris et al., 2012; HeuzéVourc'h and Courty, 2012). The aberrant expression of KLKs has led to investigations of their potential as cancer biomarkers. Several kallikrein-related peptidases, including KLK3 (PSA, prostate-specific antigen), a well-known marker for prostate cancer, have shown promise as prognostic and predictive cancer biomarkers for: cancer of the prostate, testis, kidney, breast, ovary, lung, colon, pancreas, and brain (reviewed in Schmitt and Magdolen, 2009; Avgeris et al., 2010; Kontos and Scorilas, 2012). Some KLKs have also been involved in many cancerrelated processes, such as cell growth regulation, angiogenesis, invasion, and metastasis (Sotiropoulou et al., 2009Avgeris et al., 2012). Consequently, KLKs are emerging as novel targets for pharmacological intervention (Sotiropoulou and Pampalakis, 2012). KLK6, the gene encoding kallikrein-related peptidase 6, is located within the kallikrein gene cluster on chromosome 19q13. Increased KLK6 mRNA expression has been identified in most epithelial cancers suggesting an oncogenic role for KLK6 (Bayani and Diamandis, 2011). KLK6 has also been found downregulated in breast and brain tumors (Yousef et al., 2004). KLK6 may be involved in malignant cell proliferation, as in lung and colon cancer cells (Heuze-Vourc'h et al., 2009; Kim, J. T. et al., 2011). Other findings support the implication of KLK6 in tumor invasion. KLK6 has the in vitro ability to degrade extracellular matrix proteins including laminin, fibronectin, collagens (Ghosh et al., 2004). In addition, several studies using inhibitors or siRNAs to decrease KLK6 protein expression in model cancer cell lines have revealed a concomitant reduction in the invasive potential of these cells (Shinoda et al., 2007; Henkhaus et al., 2008; Kim, J. T. et al., 2011). Conversely, KLK6 may have a protective function in breast cancer by inhibiting the epithelial-to-mesenchymal transition (Pampalakis et al., 2009). Lastly, the KLK6 gene is inactivated in metastatic breast cancer due to epigenetic events (Pampalakis and Sotiropoulou, 2006). These various findings indicate that KLK6 may exert either cancer-inhibiting or cancer-promoting activities, depending on the tissue type, stage of pathogenesis and tumor microenvironment.
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Regulation of NSCLC cell growth by KLK6 We have previously shown that elevated expression of KLK6 is an indicator of poor prognosis for patients suffering from non-small cell lung cancer (NSCLC). We used an isogenic NSCLC model cell system to demonstrate that ectopic KLK6 expression enhances cell growth by accelerating the cell cycle, between the G1 and S phases (Heuze-Vourc'h et al., 2009). We have now analyzed the way in which KLK6 triggers this response. Our data suggest that KLK6 promotes proliferation and restrains apoptosis in NSCLC cells by stimulating the EGFR signaling pathway and KLK6-dependent activation of the protease-activated receptor 2 (PAR-2).
Results KLK6 peptidase activity and cell growth and survival Ectopic KLK6 expression in cells led to increased growth of isogenic clones of A549 Flp-In (Figure 1A) and HEK293 Flp-In (Figure 1B) cells. Others have reported that proteinases regulate cell growth by mechanisms that are independent of their catalytic activity (Masson et al., 2010). We therefore evaluated the influence of KLK6 proteolytic activity on cell growth in KLK6 isogenic cell systems using a mutant KLK6 in which the serine residue essential for its enzymatic activity was replaced by an alanine residue. The stable expression of this S197A mutant KLK6 hampered KLK6-dependent cell growth (Figure 1C & 1D). Cell growth results to both cell proliferation and cell survival. As we previously reported that KLK6 regulates the growth of NSCLC cells by stimulating cell proliferation (Heuze-Vourc'h et al., 2009), we examined the capacity of this KLK to regulate the survival of A549 cells. Flow cytometry (DNA fragmentation) and ELISA (histone release) analyses revealed that ectopic KLK6 expression significantly reduced the death of staurosporine-stimulated A549 Flp-In cells (Figure 2A, B). This effect was not observed when A549 Flp-In cells were stably transduced with a vector encoding the S197A mutant (Figure 2C). These data suggest that the KLK6mediated growth of NSCLC cells is due to both proliferation and resistance to apoptosis, and is dependent on the enzymatic activity of KLK6.
KLK6 and the nuclear translocation of β-catenin Enhanced cell proliferation was reported to be associated with the down-regulation of E-cadherin and the accumulation of β-catenin in the nuclei of mouse keratinocyte cells stably
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Regulation of NSCLC cell growth by KLK6 transfected with Klk6 (Klucky et al., 2007). We used immunofluorescence analysis of controls and stable A549-KLK6 cells with an anti-β-catenin antibody to determine whether ectopic KLK6 expression modified distribution of β-catenin in NSCLC cells. β-catenin was mainly located at the cell membrane in control cells (Figure 3A) while its distribution was diffuse in A549-KLK6 cells (Figure 3B). This difference in β-catenin protein distribution was not due to altered transcription because quantitative RT-PCR revealed comparable amounts of transcripts in the control and A549-KLK6 cells (Figure 3C). Free β-catenin may be rapidly degraded, kept in the cytoplasm or translocated to the nucleus where it becomes associated with TCF/Lef family transcription factors and drives the transcription of TCF/LEF-1 target genes (Valenta et al., 2012). We therefore transfected cells with a TCF-dependent luciferase reporter plasmid (TCFwt-luci) to determine whether the ectopic KLK6 was associated with activation of β-catenin/TCF-dependent transcription. The reporter gene was twice as active in transfected A549-KLK6 cells as in control cells (Figure 3D). In contrast, no difference was measured for a luciferase reporter plasmid (TCFmut-luci) with inactive TCF-binding sites. Activation of the canonical Wnt pathway or altered E-cadherin-β-catenin association often results in the accumulation of β-catenin. This, in turn, leads to enhanced TCF/LEF-1-driven transcription. In mouse keratinocytes, β-catenin translocation induced by Klk6 follows shedding of an E-cadherin ectodomain (Klucky et al., 2007). Our Western blot analyses showed similar amounts of full-length (Figure 3E) and soluble E-cadherin (Figure 3F) in whole-cell extracts and conditioned medium from both control and A549-KLK6 cells. This suggests that KLK6 does not modify the synthesis of E-cadherin or the shedding of its ectodomain in NSCLC cells and that the translocation of β-catenin to the nucleus results from a different mechanism. Recent studies have shown that EGFR activation can also lead to the translocation of β-catenin to the nucleus in certain carcinomas (Lee et al., 2010; Ma et al., 2013). We therefore examined this pathway in NSCLC cells.
KLK6 triggers the EGFR pathway through activation of PAR-2 We treated our clones with Cetuximab to investigate whether EGFR could regulate KLK6dependent cell growth. This chimeric mouse-human antibody targets the extracellular domain of EGFR and prevents the binding of activating ligands to the EGFR. Treatment of A549 FlpIn cells with Cetuximab blocked KLK6-dependent cell growth (Figure 4A). Because EGFR signaling acts mainly through the Raf/Mek/ERK and PI3K/AKT pathways, we examined these pathways in cells that had been transiently exposed to KLK6. Addition of recombinant
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Regulation of NSCLC cell growth by KLK6 KLK6 (10 µg/ml) in the medium activated the phosphorylation of ERK in control cells (Figure 4B). This activation was inhibited by Cetuximab. Similarly, the transient expression of KLK6 stimulated the phosphorylation of Akt at Thr308 in control cells, and this activation was suppressed by cetuximab (Figure 4C). Similar results were obtained when Cetuximab was replaced by the EGFR tyrosine kinase inhibitor AG1478 (Figure 4D). As PAR2 has been described as a substrate of KLK6 (Angelo et al., 2006; Oikonomopoulou et al., 2006), we examined the influence of activating PAR2 on the ligand-dependent activation of the EGFR pathway in A549 Flp-In cells. Activation of PAR2 with an agonist peptide stimulated the growth of these NSCLC cells (Figure 5A) by a mechanism involving the binding of a ligand to EGFR. Indeed, the PAR2-dependent phosphorylation of ERK was abolished by treatment with Cetuximab (Figure 5B). We used A549 Flp-In transfected with short-interfering RNAs against PAR2 to determine whether PAR2 is involved in the response to KLK6. A nonsilencing sequence was used as a negative control. The expression of both PAR2 mRNA and protein was reduced by 90% in cells treated with PAR2-siRNA compared to that of cells treated with control-siRNA (Figure 5C). Transfection of the cells with PAR– siRNA abolished the PAR2 agonist peptide-dependent phosphorylation of ERK (Figure 5 D). Similarly, the wt.KLK6-dependent phosphorylation of ERK and Akt (Figure 6 A, B) was inhibited in the presence of a siRNA against PAR2.
Discussion KLK6 expression has been associated with a poor prognosis for patients with ovarian (Kountourakis et al., 2008; White et al., 2009; Seiz et al., 2012), gastric (Nagahara et al., 2005; Kim, J. J. et al., 2012), colon (Kim, J. T. et al., 2011) or lung cancers (Heuze-Vourc'h et al., 2009) and with glioblastoma multiforme (Drucker et al., 2013) suggesting that this protease contributes to tumor progression. The expression of KLK6 has also been connected with the increased growth and invasion of several malignant cells (Nagahara et al., 2005; Henkhaus et al., 2008; Kim, J. T. et al., 2011; Krenzer et al., 2011; Kim, J. J. et al., 2012). Our studies showed that the ectopic production of KLK6 accelerates the cell cycle, between the G1 and S phases, of NSCLC cells (Heuze-Vourc'h et al., 2009) and decreases apoptosis (described in this study). The ability of KLK6 to act on these two processes is supported by previous observations. Silencing the KLK6 gene effectively suppressed the rate at which a gastric cell line proliferated and decreased the cell population in the S phase (Nagahara et al.,
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Regulation of NSCLC cell growth by KLK6 2005). KLK6 has also been shown to enhance the survival of immune and glioblastoma cells (Scarisbrick et al., 2011; Drucker et al., 2013). Lastly, Kupferman found that KLK6 overproduction was associated with the anoikis-resistant phenotype of oral cancer cell lines (Kupferman et al., 2006). Our data indicate that a catalytically inactive mutant did not modify cell processes and signaling in the way that wild-type KLK6 did. This suggests that the active form is involved in the above processes. We believe that the zymogen secreted by the transfected cells was processed in the cell-culture medium. KLK6 was initially reported to be self-activated (Bayes et al., 2004) but subsequent studies revealed that KLK6 was essentially unable to activate proKLK6, suggesting another protease activates KLK6 (Blaber et al., 2007). The best activator of pro-KLK6 identified to date is plasmin (Yoon et al., 2008). It is conceivable that a cascade involving plasmin has generated active KLK6 in our culture conditions because the plasmin system is functional when A549 cells are cultured in the presence of serum (Van Leer et al., 2005). Both chronic and transient exposures to KLK6 modify several signaling pathways including the ERK and Akt pathways. As phosphorylation of these kinases is inhibited by silencing PAR-2, we consider that KLK6 initially signals through this G protein-coupled receptor. There is now good evidence indicating that KLKs are activators of PARs (reviewed in Oikonomopoulou et al., 2010; Caliendo et al., 2012). In particular, KLK4 has been associated with prostate cancer progression, and this effect has been linked to PAR1 and 2 (Mize et al., 2008; Ramsay et al., 2008). KLK4 signaling via PAR1 and KLK14 signaling via PAR2 have also been involved in colon tumorigenesis (Gratio et al., 2010; Gratio et al., 2011; Chung et al., 2013). Several studies have demonstrated that KLK6 can signal through PARs, mainly in the central nervous system. A KLK6-evoked intracellular Ca2+ flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes (Vandell et al., 2008). The KLK6mediated activation of PAR1 is also involved in promoting lymphocyte survival and the survival and stellation of astrocytes (Scarisbrick et al., 2011; Scarisbrick et al., 2012; Drucker et al., 2013). Finally, KLK6 plays a critical role in oligodendrogliopathy and neurodegeneration through PAR1 and PAR2 signaling (Burda et al., 2013; Yoon et al., 2013). PAR-2 stimulation has several effects on A549 cells, including the release of IL-6, IL-8 and prostaglandin E2 (PGE2), induction of COX-2 and reduction of apoptosis (Moriyuki et al., 2008; Moriyuki et al., 2009; Huang et al., 2013). PGE2 formation has been attributed to activation of the ERK/ PI3K / Akt / NF-kappaB pathway while the PAR2-triggered increase 7 / 24
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Regulation of NSCLC cell growth by KLK6 in COX-2 was described as being partly dependent on the ERK1/2-mediated activation of the beta-catenin/TCF-4 and CREB pathways (Moriyuki et al., 2008; Moriyuki et al., 2009). The signaling pathways (ERK, Akt, β-catenin/TCF) that respond to KLK6 therefore agree with those previously described in A549 cells following activation of PAR-2. Thus, these data support our belief that KLK6 drives some A549 cell functions via PAR2. As the gene c-myc is a target of TCF/LEF-1, the increase in β-catenin/TCF-dependent transcription we have observed correlates with our published data showing that KLK6 triggers c-myc induction in the same A549-FlpIn wt.KLK6 clone (Heuze-Vourc'h et al., 2009). The PAR2 agonist peptide appeared less efficient than KLK6 for promoting cell growth. This suggests that either KLK6 directly or indirectly targets other factors involved in these pathways or the canonical agonist peptide does not mimic all actions of KLK6 on PAR2. This last assumption is supported by several recent studies showing that the PARs are capable of ‘functional selectivity’ or ‘biased’ signaling (reviewed in Hollenberg et al., 2014). Darmoul and collaborators were the first to report that PAR2 could transactivate EGFR (Darmoul et al., 2004). Stimulation of PAR2 in colon cancer cells with trypsin resulted in the MMP-mediated release of an EGF family ligand (TGF- ), which in turn activated EGFR. More recent studies have reported the KLK1-triggered PAR1-dependent transactivation of EGFR in keratinocytes and neurons (Gao et al., 2009; Gao et al., 2010; Lu et al., 2014). Our results suggest that KLK6 promotes the PAR-2-dependent transactivation of EGFR. Both Cetuximab and AG1478, two inhibitors of the EGFR pathways, blocked the KLK6dependent phosphorylation of ERK and Akt. The inhibition by Cetuximab indicates that transactivation of the EGFR pathway is probably ligand-dependent. Further studies are required to identify the molecules involved in transmission of the signal between PAR-2 and EGFR. This process usually implicates members of the disintegrin and metalloproteinase (ADAM) family that perform the shedding of the ectodomain of membrane-bound EGFR ligands such as TGF-α (transforming growth factor-α), HB-EGF (heparin-binding EGF-like growth factor) and amphiregulin (Kataoka, 2009). A second mechanism for transactivation of the EGFR by PAR2 signaling has been described in some circumstances {Caruso et al., 2006; van der Merwe et al., 2008}. Stimulation of PAR2 may cause activation of Src which in turn leads, via an intracellular route, to the tyrosine phosphorylation and subsequent activation of the EGFR {Caruso, 2006 #2362; van der Merwe, 2008 #2361}. Whether this alternative pathway is involved in the KLK6-dependent transactivation of the EGFR remains to be determined.
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Regulation of NSCLC cell growth by KLK6 In summary, our results suggest that KLK6 promotes the proliferation of NSCLC cells and restricts their apoptosis via an activation cascade initiated by PAR2 and involving the liganddependent transactivation of EGFR.
Materials and methods Materials HEK-293 Flp-In cells, cell culture media, antibiotics, Lipofectamine, cloning plasmids, SuperScript VILO cDNA kit and Alexa Fluor 488 were all from Invitrogen (Fisher Bioblock Scientific, Illkirch, France). Parental A549 cells were from the American Type Culture Collection. Fetal calf serum (FCS) was from Lonza (Verviers, Belgium). TOP-flash plasmid DNA and negative control FOP-flash plasmid DNA were purchased from Addgene (Cambridge, USA). SYBR® Premix Ex TaqTM kit and siRNA PAR2 were from Takara (Ozyme, St-Quentin en Yvelines, France) and Applied Biosystems (Courtaboeuf, France), respectively. CellTiter 96® Aqueous One Solution Cell Proliferation Assay and DualLuciferase Assay System were from Promega France (Charbonnieres les Bains, France). Cell Death Detection ELISA kit was from Roche Molecular Biochemicals (Meylan, France). AntissDNA/APOSTAIN F7-26 monoclonal antibody was from Eurobio-Abcys (Courtaboeuf, France). FITC-conjugated anti-mouse IgM antibody was from Sigma-Aldrich (Saint-Quentin Fallavier, France). Anti-β-catenin, Anti- soluble E-cadherin, phospho-Erk1/2 and total Erk1/ 2 antibodies were from Santa-Cruz Biotechnology (CliniSciences, Nanterre, France). Antiphospho-Akt (Thr 308; Ser 473) and anti-Akt were from Cell Signalling Technology (Ozyme, France). Cetuximab/Erbitux was from Merck Serono (Lyon, France). Other reagents were from Sigma-Aldrich. Recombinant KLK6 was produced, purified and activated as previously described (Oikonomopoulou et al., 2008).
Culture conditions and cell viability assay A549 Flp-In cells and the A549 Flp-In clone expressing wt.KLK6 were previously described (Heuze-Vourc'h et al., 2009). The A549 Flp-In cells were grown in RPMI-1640 Glutamax and the HEK293 Flp-In cells were grown in D-MEM. The media were supplemented with 10% FCS, 2mM L-glutamine, 100U/ml penicillin and either 100 µg/ml zeocin or 100 µg/ml hygromycin. The cells used in the viability assay were seeded in 96-well plate and maintained
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Regulation of NSCLC cell growth by KLK6 at 37 °C for an appropriate time. Medium was then removed and cells were incubated for 2 h at 37 °C in 100µl culture medium and 20µl CellTiter 96® Aqueous One Solution Cell Proliferation reagent containing MTS. The absorbance at 490 nm was determined with a microplate reader.
Apoptosis analysis Cells were seeded in 96-well plate (ELISA) at 5×103 cells per well or in six-well plates (APOSTAIN) at 5×105 cells per well and incubated for 16 hours. The medium was then collected and the cells incubated in RPMI, 2% FCS with 0.2 µM staurosporine for 24h. Apoptosis was measured with the Cell Death Detection ELISA kit in accordance with the manufacturer's instructions. The cells used in the APOSTAIN analysis were fixed with icecold methanol and collected by centrifugation. They were then suspended in 250 μl formamide for 5 min at room temperature and incubated at 75°C for 10 min. Then, 2 ml of PBS containing 1% of nonfat dried milk were added and cells were incubated for 15 min at room temperature, collected by centrifugation and incubated for 15 min in 75 μl PBS containing 5% FCS and anti-ssDNA F7-26 monoclonal antibody. These cells recovered by centrifugation and incubated for 15 min in 100 μl PBS containing FITC-conjugated antimouse IgM (20 μg/ml) and 1% nonfat dried milk. Cells were rinsed, centrifuged, and resuspended in 200 μl of PBS containing 1 μg/mL of 7-AAD. Negative controls were treated with IgM instead of the specific primary antibody. All cells were then analyzed by flow cytometry.
Immunofluorescence analysis A549 cells were grown to 90% confluence in LAB-TEK chamber slides, fixed with ice-cold methanol for 10 minutes and wash repeatedly with PBS (pH 7.2). These fixed cells were incubated for 30 min with blocking buffer (PBS plus 1% bovine serum albumin (BSA), then for 2 h with anti-β-catenin antibody diluted to 4µg/ml in incubation buffer (1% BSA in PBS) and washed thoroughly with PBS. Finally, they were incubated for 1 h with Alexa Fluor 488 diluted in incubation buffer, washed
with PBS, and mounted with mounting medium
(IF fluoromount G) on glass slides. Images were acquired by confocal microscopy.
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Regulation of NSCLC cell growth by KLK6
Isolation of total RNA and real-time PCR analysis Total RNA was extracted using Total ARN Isolation Nucleospin RNA II kits according to the manufacturer‘s instructions (Macherey-Nagel, Hoerdt, France). The eluted RNA was quantified by spectrophotometry and 5 μg aliquots were reverse-transcribed to obtain the corresponding cDNAs with SuperScript VILO cDNA kits. Real-time quantitative PCR was performed with SYBR® Premix Ex TaqTM kits using the Roche LightCycler 480 instrument. The primer sets were: for β-catenin 5′-CAC AAG CAG AGT CCA AAG ACA G-3′ and 5′GAT TCC TGA GAG TCC AAA GAC AG-3′, for β-actin 5′-GAC CAT TGG CAA TGA GCG GTT C-3′ and 5′-AGG TCT TTG CGG ATG TCC ACG T-3′, and for PAR2 5’-CTC CTC TCT GTC ATC TGG TTC C-3’ and 5’-TGC ACA CTG AGG CAG GTC ATG A-3’.
Luciferase reporter assay A549 Flp-In and A549-KLK6 cells were seeded in 24-well plates and incubated for 16h as above. They were then transfected with the TOP-flash plasmid DNA (TCFwt) or the negative control FOP-flash plasmid DNA (TCFmut) together with Renilla luciferase-coding plasmids by incubation for 4 h with Lipofectamine Plus TM in serum and antibiotic-free OptiMEM. The medium was changed to RPMI plus antibiotics and 10% FCS and the cells grown for a further 16 h. The Firefly and Renilla luciferase activities in these cells were measured using the Dual-Luciferase Assay System according to the manufacturer’s instructions. Firefly luciferase was normalized to Renilla luciferase activity.
Western blot analysis Whole cell extracts were prepared with lysis buffer A (200 mM Tris pH8, 137 mM NaCl, 1% NP40, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, proteinase inhibitor cocktail). Equal amounts of protein (cell lysates or supernatants) were loaded onto SDSpolyacrylamide gels, separated, and the resulting proteins transferred to polyvinylidene fluoride membranes, whose free sites had been blocked by incubation for 1 h at room temperature with TBS Tween-20 buffer containing 5% dried milk (soluble E-cadherin and phospho-Erk1/2) or 1% BSA (total Erk1/2). The membranes were then incubated with primary antibodies [anti-soluble E-cadherin (1:1000), anti-phospho-Erk1/2 (1:500), anti-total Erk1/2 (1:1000)] overnight at 4°C. The blots were incubated with horseradish peroxidase (HRP) labeled anti-mouse immunoglobulin G or anti-rabbit immunoglobulin G (1:5000) at
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Regulation of NSCLC cell growth by KLK6 room temperature for 2 hrs in TBS Tween-20 containing 5% dried milk. Proteins were revealed by enhanced chemiluminescence.
siRNA transfection A549 cells (0.3×106 cells / well in 6-well plates) were incubated overnight and then transfected by incubation with 12.5 pmol of a small interfering RNA (siRNA) that targeted PAR2 transcripts in OptiMEM plus Lipofectamine for 4 h. Control transfections were performed with an nonsilencing control siRNA. The transfected cells were washed once with PBS and then incubated for 24 h in RPMI plus 10% FCS. The medium was changed and the cells were treated with cetuximab and/or PAR2 agonist.
Statistics All statistical analyses were performed with GraphPad Prism 5.00 (GraphPad Software Inc., San Diego, CA, USA). Results are given as means ± SEM of at least 3 independent experiments.
Acknowledgements The English text was edited by Dr. Owen Parkes. Financial support was provided by the Ligue Contre le Cancer (Comités d’Indre et d’Eure et Loire) and Région Centre (grant KalliCap). Noémie Michel was the recipient of a Fellowship from the Région Centre.
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Regulation of NSCLC cell growth by KLK6
References Angelo, P. F., Lima, A. R., Alves, F. M., Blaber, S. I., Scarisbrick, I. A., Blaber, M., Juliano, L., and Juliano, M. A. (2006). Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects. J. Biol. Chem. 281, 3116-3126. Avgeris, M., Mavridis, K., and Scorilas, A. (2010). Kallikrein-related peptidase genes as promising biomarkers for prognosis and monitoring of human malignancies. Biol. Chem. 391, 505-511. Avgeris, M., Mavridis, K., and Scorilas, A. (2012). Kallikrein-related peptidases in prostate, breast, and ovarian cancers: from pathobiology to clinical relevance. Biol. Chem. 393, 301317. Bayani, J., and Diamandis, E. P. (2011). The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6). Clin. Chem. Lab. Med. 50, 211-233. Bayes, A., Tsetsenis, T., Ventura, S., Vendrell, J., Aviles, F. X., and Sotiropoulou, G. (2004). Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation. Biol. Chem. 385, 517-524. Blaber, S. I., Yoon, H., Scarisbrick, I. A., Juliano, M. A., and Blaber, M. (2007). The autolytic regulation of human kallikrein-related peptidase 6. Biochemistry 46, 5209-5217. Burda, J. E., Radulovic, M., Yoon, H., and Scarisbrick, I. A. (2013). Critical role for PAR1 in kallikrein 6-mediated oligodendrogliopathy. Glia 61, 1456-1470. Caliendo, G., Santagada, V., Perissutti, E., Severino, B., Fiorino, F., Frecentese, F., and Juliano, L. (2012). Kallikrein Protease Activated Receptor (PAR) Axis: An Attractive Target for Drug Development. J. Med. Chem. 55, 6669-6686. Caruso, R., Pallone, F., Fina, D., Gioia, V., Peluso, I., Caprioli, F., Stolfi, C., Perfetti, A., Spagnoli, L. G., Palmieri, G., et al. (2006). Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation. Am J Pathol. 169, 268-278. Chung, H., Ramachandran, R., Hollenberg, M. D., and Muruve, D. A. (2013). Proteinaseactivated Receptor-2 Transactivation of Epidermal Growth Factor Receptor and Transforming Growth Factor-beta Receptor Signaling Pathways Contributes to Renal Fibrosis. J. Biol. Chem. 288, 37319-37331. Darmoul, D., Gratio, V., Devaud, H., and Laburthe, M. (2004). Protease-activated receptor 2 in colon cancer: trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation. J. Biol. Chem. 279, 20927-20934 Drucker, K. L., Paulsen, A. R., Giannini, C., Decker, P. A., Blaber, S. I., Blaber, M., Uhm, J. H., O'Neill, B. P., Jenkins, R. B., and Scarisbrick, I. A. (2013). Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma. Neurol. Oncol. 15, 305-318. Gao, L., Chao, L., and Chao, J. (2009). A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor. Exp. Cell Res. 316, 376-389. Gao, L., Chao, L., and Chao, J. (2010). A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor. Exp. Cell Res. 316, 376-389.
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Regulation of NSCLC cell growth by KLK6 Ghosh, M. C., Grass, L., Soosaipillai, A., Sotiropoulou, G., and Diamandis, E. P. (2004). Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol. 25, 193-199. Gratio, V., Beaufort, N., Seiz, L., Maier, J., Virca, G. D., Debela, M., Grebenchtchikov, N., Magdolen, V., and Darmoul, D. (2010). Kallikrein-related peptidase 4: a new activator of the aberrantly expressed protease-activated receptor 1 in colon cancer cells. Am. J. Pathol. 176, 1452-1461. Gratio, V., Loriot, C., Duke Virca, G., Oikonomopoulou, K., Walker, F., Diamandis, E. P., Hollenberg, M. D., and Darmoul, D. (2011). Kallikrein-Related Peptidase 14 Acts on Proteinase-Activated Receptor 2 to Induce Signaling Pathway in Colon Cancer Cells. Am. J. Pathol. 179, 2625-2636. Henkhaus, R. S., Gerner, E. W., and Ignatenko, N. A. (2008). Kallikrein 6 is a mediator of KRAS-dependent migration of colon carcinoma cells. Biol. Chem. 389, 757-764. Heuzé-Vourc'h, N., and Courty, Y. (2012). Pathophysiology of Kallikrein-related Peptidase in Lung Cancer. In: Kallikrein-Related Peptidases, V. Magdolen, C.P. Sommerhoff, H. Fritz, and M. Schmitt, eds. (De Gruyter), pp. 3-26. Heuze-Vourc'h, N., Planque, C., Guyetant, S., Coco, C., Brillet, B., Blechet, C., Parent, C., Briollais, L., Reverdiau, P., Jourdan, M. L., and Courty, Y. (2009). High Kallikrein-Related Peptidase 6 in Non-Small Cell Lung Cancer Cells: an indicator of tumor proliferation and poor prognosis. J. Cell. Mol. Med. 13, 4014-4022. Huang, S. H., Li, Y., Chen, H. G., Rong, J., and Ye, S. (2013). Activation of proteinaseactivated receptor 2 prevents apoptosis of lung cancer cells. Cancer Invest. 31, 578-581. Kataoka, H. (2009). EGFR ligands and their signaling scissors, ADAMs, as new molecular targets for anticancer treatments. J. Dermatol. Sci. 56, 148-153. Hollenberg, M. D., Mihara, K., Polley, D., Suen, J. Y., Han, A., Fairlie, D. P., and Ramachandran, R. (2014). Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease. Br. J. Pharmacol. 171, 1180-1194. Kim, J. J., Kim, J. T., Yoon, H. R., Kang, M. A., Kim, J. H., Lee, Y. H., Kim, J. W., Lee, S. J., Song, E. Y., Myung, P. K., and Lee, H. G. (2012). Upregulation and secretion of kallikreinrelated peptidase 6 (KLK6) in gastric cancer. Tumour Biol. 33, 731-738. Kim, J. T., Song, E. Y., Chung, K. S., Kang, M. A., Kim, J. W., Kim, S. J., Yeom, Y. I., Kim, J. H., Kim, K. H., and Lee, H. G. (2011). Up-regulation and clinical significance of serine protease kallikrein 6 in colon cancer. Cancer 117, 2608-2619 Klucky, B., Mueller, R., Vogt, I., Teurich, S., Hartenstein, B., Breuhahn, K., Flechtenmacher, C., Angel, P., and Hess, J. (2007). Kallikrein 6 induces E-cadherin shedding and promotes cell proliferation, migration, and invasion. Cancer Res. 67, 8198-8206. Kontos, C. K., and Scorilas, A. (2012). Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers. Clin. Chem. Lab. Med. 50, 1877-1891. Kountourakis, P., Psyrri, A., Scorilas, A., Camp, R., Markakis, S., Kowalski, D., Diamandis, E. P., and Dimopoulos, M. A. (2008). Prognostic value of kallikrein-related peptidase 6 protein expression levels in advanced ovarian cancer evaluated by automated quantitative analysis (AQUA). Cancer Sci. 99, 2224-2229.
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Regulation of NSCLC cell growth by KLK6
Figures
Figure 1
Active KLK6 promotes cell growth, while the mutant KLK6 form does not.
(A and B) The growth of parental Flp-In cells (dashed lines) and isogenic clones stably producing wild-type KLK6 (full lines) was evaluated by counting cells at the indicated times; (A) A549 Flp-In cells, (B) HEK293 Flp-In cells. (C) A549 and (D) HEK-293 cells stably producing wild-type KLK6 (wt.KLK6) or the S197A mutant (mut.KLK6) were seeded at the same density and cell growth was measured after 48h using an MTS proliferation assay. Values are means ± SEM. Krustal-Wallis test, *P
