- Open Access
Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients
© Schrader et al.; licensee BioMed Central. 2015
- Received: 10 November 2014
- Accepted: 8 May 2015
- Published: 20 May 2015
Dysregulated expression of Kallikrein-related peptidase 6 (KLK6) is a common feature for many human malignancies and numerous studies evaluated KLK6 as a promising biomarker for early diagnosis or unfavorable prognosis. However, the expression of KLK6 in carcinomas derived from mucosal epithelia, including head and neck squamous cell carcinoma (HNSCC), and its mode of action has not been addressed so far.
Stable clones of human mucosal tumor cell lines were generated with shRNA-mediated silencing or ectopic overexpression to characterize the impact of KLK6 on tumor relevant processes in vitro. Tissue microarrays with primary HNSCC samples from a retrospective patient cohort (n = 162) were stained by immunohistochemistry and the correlation between KLK6 staining and survival was addressed by univariate Kaplan-Meier and multivariate Cox proportional hazard model analysis.
KLK6 expression was detected in head and neck tumor cell lines (FaDu, Cal27 and SCC25), but not in HeLa cervix carcinoma cells. Silencing in FaDu cells and ectopic expression in HeLa cells unraveled an inhibitory function of KLK6 on tumor cell proliferation and mobility. FaDu clones with silenced KLK6 expression displayed molecular features resembling epithelial-to-mesenchymal transition, nuclear β-catenin accumulation and higher resistance against irradiation. Low KLK6 protein expression in primary tumors from oropharyngeal and laryngeal SCC patients was significantly correlated with poor progression-free (p = 0.001) and overall survival (p < 0.0005), and served as an independent risk factor for unfavorable clinical outcome.
In summary, detection of low KLK6 expression in primary tumors represents a promising tool to stratify HNSCC patients with high risk for treatment failure. These patients might benefit from restoration of KLK6 expression or pharmacological targeting of signaling pathways implicated in EMT.
The Kallikrein-related peptidase 6 (KLK6) belongs to a family of 15 secreted serine proteases with trypsin or chymotrypsin-like activity . KLKs are encoded by a cluster of genes located on human chromosome 19q13.3–13.4 . Deregulation of KLK6 expression occurs in neurodegenerative and skin disorders, and is a common event in human cancer (reviewed in [3, 4]). Most of these cancers, such as glioma, ovarian, breast, uterine, pancreatic, colorectal, gastric, skin, urinary bladder, lung and salivary gland tumors, were characterized by a strong increase of KLK6 transcript and protein levels as compared to normal tissues (reviewed in [1, 5, 6]). Consequently, KLK6 represents a promising biomarker of early diagnosis and/or unfavorable prognosis in several human malignancies. Additionally, KLK6 expression was found in tumor adjacent epithelial and stromal cells, and may considerably contribute to the aggressiveness of cancer cells by a paracrine mode of action [7, 8].
Despite this overwhelming evidence pointing to a critical role of KLK6 in neoplastic transformation and malignant progression, the regulation and function of KLK6 under physiological and pathological settings is poorly understood. The regulation of KLK6 expression under pathological conditions is most likely influenced by multiple mechanisms, including gene copy number imbalances [9, 10], exposure to steroid hormones [11, 12], epigenetic events such as gene promoter hyper-methylation [13–15], oncogenic signaling [16–19], and posttranscriptional control by miRNAs [20, 21].
KLK6 can degrade components of the extracellular matrix in vitro and participates in cellular processes such as inflammation, receptor activation, and regulation of apoptosis (reviewed in [1, 3, 5]). Moreover, KLK6 is implicated in tissue remodeling and induction of tumor-relevant processes such as proliferation, migration and invasion (reviewed in [1, 3]). However, recent studies questioned the general oncogenic role of KLK6 and stressed the importance to consider its context-dependent, tumor-protective function, as exemplified in breast and renal cancer [14, 22–25].
So far, the expression of KLK6 in head and neck squamous cell carcinoma (HNSCC) and its association with pathological features or the clinical outcome has not been addressed in larger patient cohorts. HNSCC arise from mucosal epithelia lining of the upper aero-digestive tract and represent one of the most common and lethal human cancers worldwide [26, 27]. While tobacco and alcohol consumption remain the major risk factors, more recent findings have established infection by high-risk human papilloma viruses, especially HPV16, as an important cause for a subgroup of HNSCC [28, 29]. Implementation of intensified and multimodal treatment has improved the clinical outcome of HNSCC, but often causes severe toxicity and debilitating long-term impacts on quality of life accompanied with only limited clinical benefit. Accordingly, only 40-50 % of patients with an advanced disease will survive for five years after primary treatment , and appropriate therapy of advanced HNSCC still remains a major challenge. Therefore, prognostic biomarkers are urgently needed for better stratification of patients with high risk for treatment failure, and to support the identification of novel drug targets for more efficient and less toxic therapies.
In the current study, we conducted loss-of-function and gain-of-function approaches in mucosal tumor cell lines to investigate the contribution of KLK6 in the regulation of tumor development and malignant progression. We demonstrate that silencing of KLK6 expression promotes tumor cell proliferation, migration and invasion in vitro. This phenotype is accompanied by the induction of epithelial-to-mesenchymal transition (EMT), nuclear accumulation of β-catenin and resistance against irradiation. The clinical relevance of our findings is supported by the fact that low KLK6 protein level in primary HNSCCs serves as unfavorable risk factor for progression-free and overall survival.
KLK6 regulates tumor cell proliferation
FaDu-shKLK6 clones were characterized by a significant increase in the total number of cells over a time period of six days (Fig. 1C). The degree of the phenotype was associated with the silencing efficiency and less prominent for FaDu-shKLK6#1. A decrease in tumor cell growth was consistent with reduced BrdU incorporation in FaDu-Mock as compared to FaDu-shKLK6 clones (Fig. 1D).
To further support our findings on KLK6-mediated growth inhibition, we transfected KLK6-negative HeLa cells with control (HeLa-Mock) or pcDNA-KLK6-Myc/His plasmids (HeLa-KLK6) to generate stable clones with ectopic KLK6 overexpression (Additional file 1: Fig. S1A-B). Indeed, HeLa-KLK6 clones had a significantly reduced tumor cell growth in comparison to HeLa-Mock controls (Additional file 1: Fig. S1C).
KLK6 regulates tumor cell morphology, migration and invasion
KLK6 expression and epithelial-to-mesenchymal transition
Next, we investigated the expression of well-established regulators of EMT by quantitative RT-PCR with cDNA derived from FaDu-Mock and FaDu-shKLK6 clones. In addition to significantly induced Vimentin (VIM) and reduced E-cadherin (CDH1) transcript levels FaDu-shKLK6 clones showed higher transcript levels for SNAIL1 (SNAI1), ZEB1, ZEB2, Fibronectin (FN1), and N-cadherin (CDH2) (Fig. 3C). In contrast, TWIST1 and SLUG (SNAI2) transcript levels were reduced by KLK6 silencing.
Loss of KLK6 expression induces nuclear β-catenin accumulation
KLK6 expression and radiosensitivity
An increasing body of experimental evidence supports that an EMT-like phenotype is related to treatment resistance of tumor cells. To investigate whether silencing of KLK6 alters the radiosensitivity of FaDu cells, we performed a colony-forming assay. In the absence of irradiation FaDu-shKLK6 clones developed larger colonies as compared to mock controls, which was consistent with an increase in proliferation (Fig. 4D). In addition, the total number of colonies was higher for FaDu-shKLK6 clones, which might be due to an increased percentage of cells with self-renewal capacity as a consequence of Wnt/β-catenin activity. After irradiation with single doses of 2 and 5 Gy, respectively, the relative survival fraction was significantly higher in FaDu cells with silenced KLK6 expression (Fig. 4E), suggesting a higher radiosensitivity of mock controls.
KLK6 protein levels in human oropharyngeal and laryngeal SCCs
Correlation analysis for KLK6 protein expression and clinico-pathological features of the combined HNSCC cohort
Lymph node status
Multivariate analysis of overall and progression-free survival for HNSCC patients of the combined cohort
HR (95 % CI)
HR (95 % CI)
low vs. high1
<58.621 vs. ≥58.62
male1 vs. female
I/II1 vs. III/IV
G1/21 vs. G3
non-smoker1 vs. smoker2
non-drinker1 vs. drinker3
In summary, these data demonstrate a close association between loss of KLK6 expression and worse clinical outcome of LSCC and OPSCC patients, and strongly support the assumption that KLK6 might serve as a reliable prognostic biomarker to identify HNSCC patients at high risk for treatment failure.
Aberrant expression of KLK6 is a common feature for many human malignancies and numerous studies evaluated KLK6 as a prognostic biomarker (reviewed in ). Consistent with most reports, we found high KLK6 expression in primary tumor samples of 42.6 % HNSCC patients. However, while induced KLK6 expression alone or in combination with other KLK family members was associated with poor progression-free and overall survival in colorectal cancer [31, 32], gastric cancer [33, 34], pancreatic ductal adenocarcinoma , ovarian cancer [36, 37], lung cancer , and intracranial tumors [39, 40], a high KLK6 expression pattern served as favorable prognostic biomarker in our OPSCC and LSCC patient cohorts. These data suggest a context-specific role of KLK6 in regulating the malignant progression and response to therapy, with both tumor promoting and tumor protective functions depending on the cellular origin of the tumor tissue. However, it is worth noting that the tumor promoting function of KLK6 in most human cancers has been deduced from the association of its expression and clinical or pathological features. But only few studies address the underlying molecular mechanisms in vitro, and confirmations in preclinical model systems are missing.
So far, only two studies investigated the prognostic value of other KLKs in laryngeal cancer, while to the best of our knowledge no study was published on oropharyngeal SCCs. Both studies reported a remarkable down-regulation of either KLK4 or KLK11 transcript levels in laryngeal cancer as compared to their non-malignant counterparts [41, 42]. Similar to our data on KLK6, patients with KLK11-positive tumors had a favorable prognosis , and low KLK4 expression predicted short-term relapse and poor disease-free survival . Finally, down-regulation of KLK13 was reported in oral SCC cell lines and low KLK13 expression in primary oral cancer significantly correlated with regional lymph node metastasis [43, 44].
A tumor protective role of KLK6 was demonstrated in breast cancer, where it was identified originally as a putative tumor suppressor due to its down-regulation during metastasis . Tumor-specific loss of KLK6 expression in breast cancer cells is mediated by epigenetic silencing initiated by hyper-methylation at the proximal promoter . It will be interesting to address whether a similar mode of regulation also occurs in primary HNSCC with low KLK6 expression, and in local recurrence or metastasis that develop in HNSCC patients with treatment failure.
Pampalakis and colleagues demonstrated that restoration of physiological KLK6 levels in breast cancer cell lines reverted the malignant phenotype in vitro and in vivo . They provided compelling evidence that KLK6 may act as suppressor for tumor progression by promoting a mesenchymal-to-epithelial transition . In line with our findings in HeLa cells, reactivation of KLK6 in breast cancer cells was associated with prominent down-regulation of Vimentin in the absence of a parallel rise in E-cadherin (unpublished data). These data suggest a common inhibitory function of KLK6 on Vimentin expression in breast and mucosal epithelial cells; however, the molecular mechanism remains to be fully elucidated. Moreover, silencing of KLK6 in a HNSCC cell line with prominent KLK6 expression resulted in an EMT-like phenotype with strong up-regulation of Vimentin, complete loss of E-cadherin and deregulation of other well-established molecular markers of EMT. On the cellular level loss of KLK6 was associated with accelerated migration and invasion as well as impaired response to irradiation, well-known characteristics of EMT and most likely responsible for the poor clinical outcome of HNSCC patients with low KLK6 expression. Our data are in contrast to recent findings in other tumor cells lines derived from skin and colon cancer in which KLK6 promotes tumor cell migration and invasion by either E-cadherin ectodomain shedding , activation of protease-activated receptors  or so far unknown molecular mechanisms . Although the principle nature for these cell type specific differences remains elusive, Pampalakis and colleagues claimed that KLK6 regulates EMT in breast cancer cells via the TGF-β pathway [14, 47]. However, neither FaDu cells with silenced KLK6 expression nor HeLa cells with ectopic overexpression exhibited obvious changes in SMAD2/3 phosphorylation, questioning a major impact of KLK6 on canonical TGF-β signaling, at least in mucosal tumor cells. Intriguingly, our study demonstrated nuclear accumulation of β-catenin and activation of TCF-dependent gene expression upon KLK6 silencing in FaDu cells. Nuclear accumulation of β-catenin was also evident in KLK6low tumor samples of HNSCC patients. Intracellular expression of β-catenin has been reported in the context of invasive growth and metastasis of oral carcinoma cells as well as poor prognosis . β-catenin is a component of the cell-cell adhesion complex and anchored to cadherin-related proteins, which sequester β-catenin at the cell periphery. Consequently, nuclear accumulation of β-catenin might result from down-regulation of E-cadherin during EMT after silencing of KLK6 in vitro or loss of E-cadherin expression in KLK6low tumor cells during malignant progression of HNSCCs. In addition, β-catenin is also a key regulator of the Wnt signaling. Wnt/β-catenin is one of the crucial pathways in the maintenance of the self-renewal capacity of stem cells, and its inhibition is a promising approach to target cancer stem cells from HNSCC [49, 50]. In contrast to our data, KLK6-induced nuclear translocation of β-catenin was demonstrated in mouse keratinocytes and human lung cancer cell lines in vitro [45, 51], further supporting a diverse and context dependent function of KLK6 in malignant progression.
Cell culture experiments
Human HNSCC cell lines (FaDu, Cal27 and SCC25 purchased from ATCC) and the cervix carcinoma cell line HeLa were maintained in Dulbecco’s Modified Eagle’s Medium (Sigma, Germany) supplemented with 10 % fetal bovine serum (Sigma, Germany), 2 mM L-Glutamine (Sigma, Germany) and 50 μg/ml Penicillin-Streptomycin (Sigma, Germany) in a humidified atmosphere of 6-8 % CO2 at 37 °C. Authentication of all cell lines was confirmed by the Multiplex Human Cell Line Authentication Test (Multiplexion, Germany).
FaDu cells were transfected with either a control pRS vector encoding a non-effective Hush 29-mer scrambled shRNA cassette (TR30012, OriGene Technologies, USA) or a pRS-shKLK6 plasmid (TR316673, OriGene Technologies, USA) using FuGene HD Transfection Reagent (Promega, Germany) according to the manufacturer’s instruction. Following selection with 1.5 μg/ml Puromycin (Gibco life technologies, Germany) for one week, stable clones were isolated and KLK6 silencing was confirmed on RNA level. HeLa cells were transfected with either the pcDNA3.1-Myc/His control or pcDNA3.1-KLK6-Myc/His expression plasmids using FuGene HD Transfection Reagent (Promega, Germany) according to the manufacturer’s instruction. Cloning of pcDNA-KLK6-Myc/His plasmid was described previously . Following selection with 0.4 mg/ml Neomycin (Calbiochem Merck, Germany) for two weeks, stable single clones were isolated and ectopic KLK6 expression was confirmed on RNA and protein level.
Tumor cell growth and proliferation
1,000 cells per well were seeded in 12-well plates. At the indicated time points after seeding, cells were trypsinized, stained with trypan blue and total number of living cells was determined with the help of a Neubauer counting chamber. For the BrdU cell proliferation assay, 20,000 cells per well were seeded on glass coverslips in 12-well plates. 48 hours after seeding, cells were treated for 30 minutes with 1 mM BrdU, washed three times with 1xPBS for five minutes, and fixed for seven minutes with cold methanol (-20 °C). After intense washing with 1xPBS, fixed cells were treated for 30 minutes at 37 °C with 2M HCl, followed by incubation in borate buffer for 10 minutes. After intense washing with 1xPBS, samples were incubated for 30 minutes at RT with 10 % goat serum in 0,1 % Triton-X-100 in PBS. Next, samples were incubated overnight and at 4 °C with the Alexa555-conjugated BrdU-antibody (diluted 1:100 in PBS). After intense washing with 1xPBS stained cells were incubated with Hoechst 33342 (Calbiochem Merck, Germany) for five minutes and mounted with Mowiol.
Phalloidin and immunofluorescence staining
20,000 cells per well were seeded on glass coverslips in a 12-well plate. 48 hours after seeding, cells were fixed with 4 % PFA for 15 minutes, treated for 30 minutes with 0.5 % Triton-X100 in PBS, and incubated for 30 minutes with 0.2 % Tween 20/1 % BSA in TBS. Following incubation for 1–2 hours at RT with the first antibody diluted in 0.2 % Tween/1 %BSA in TBS, the secondary antibody diluted in 0.2 % Tween/1 % BSA in TBS was added. After incubation for 30–60 minutes at RT, nuclei were visualized by incubation of cells with Hoechst 33342 (Calbiochem Merck, Germany) and cytoskeleton rearrangement was visualized by incubation with Phalloidin-Alexa Fluor 488 (Invitrogen, Germany) as described previously . Finally coverslips were mounted with Mowiol. Reagents and antibodies used are listed in Additional file 5: Table S2.
Migration and invasion assay
To assess tumor cell migration, 21,000 cells were seeded into each chamber of sterile culture inserts (ibidi, Germany), which were placed in 6-well plates. 12 hours after seeding, cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany) in the incubator and insert were removed. Pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02 at the indicated time points and analyzed with the software tool ImageJ to quantify the kinetic of gap closure over time.
To determine tumor cell invasion, FaDu-shKLK6 and FaDu-Mock cells were treated for one hour with 10 μg/ml Mitomycin C (Sigma, Germany), trypsinized and 100,000 cells were seeded in matrigel-coated Boyden chamber inserts (BD Bioscience, Germany), which were placed in 12-well plates. After 48 hours, non-invading cells were removed using cotton swaps and remaining cells were fixed for ten minutes in 4 % PFA and stained with Hoechst 33342 (Calbiochem Merck, Germany). Following mounting with Mowiol, five representative pictures were taken with the Nikon Eclipse Ti microscope using the Nikon Imaging Software NIS-Elements 3.20.02, and the amount of invaded cells was counted with the software tool ImageJ.
Colony forming assay
To determine the response to irradiation, 100, 300 and 1,000 cells were seeded per well in 6-well plates and irradiated at a dose of 2 or 5 gray (Gy) using X-RAD 320 (Precision X-Ray, North Branford, CT USA) or kept untreated as controls. After 9 days in culture, clones were stained with crystal violet and total number of colonies was quantified as described in . The survival fraction was computed according to .
RNA extraction, cDNA synthesis and RT-PCR analysis
Total RNA from tumor cell lines was isolated with the RNeasy Mini Kit (Qiagen, Germany) following the manufacturer’s instruction. DNA digestion was performed with RNase-free DNAse Set (Qiagen, Germany). Quantity and quality of isolated RNA was determined with the help of the Nanodrop Spectrophotometer ND-1000 (peqlab, Germany). For cDNA synthesis 5 μg total RNA were diluted in 35 μl nuclease-free H2O. 0.5 μl Oligo (dT) primers were added and incubated for five minutes at 70 °C. A mixture of 10 μl 5xRevertAid Buffer, 2 μl 25 mM dNTP mix, 2 μl Ribolock RNase Inhibitor and 0.5 μl RevertAid Reverse Transcriptase (all from Fermentas, Germany) was added per reaction and incubated for one hour at 42 °C. For semi-quantitative RT-PCR a ready-to-use mix (Red Load Taq Master, Jena Bioscience, Germany) was used according to manufacturer’s instructions. Amplicons were separated by 1 % agarose gel electrophoresis and visualized with GelRed (Biotium, USA) using the UV documentation system for agarose gel (peqlab, Germany). Quantitative RT-PCR was performed as described previously . As reference genes transcript levels of ACTB, LMNB1 and TBP were quantified. Specific annealing temperatures and sequences of all primers are listed in Additional file 5: Table S1. The cycle of threshold (CT) of the gene of interest was standardized to the CT values of the reference genes (ACTB, LMNB and TBP) using the ∆∆CT method. For each primer pair, primer efficiency was tested by a dilution series from 0.01 to 100 ng of a cDNA mix of all samples. A primer efficiency of 1.8 to 2.0 was accepted for further analysis.
Western blot analysis
Western blot analysis was performed with whole cell lysates or enriched protein fractions from cell culture supernatants as described previously . Antibodies and dilutions that were used for Western blot analysis are listed in Additional file 5: Table S2.
Luciferase reporter assay
30,000 FaDu-Mock and FaDu-shKLK6 cells were seeded in 24-well plates and transfected with either 0.3 μg TOPFLASH or 0.3 μg FOPFLASH (upstate biotechnology, USA) using FuGene HD Transfection Reagent (Promega, Germany) according to the manufacturer’s instruction. As reference for transfection efficiency all cells were co-transfected with 0.3 μg pTK-RL (Promega, Germany). Firefly and Renilla luciferase activity was quantified with a Sirius Luminometer (Berthold Detection Systems, Germany) using the Dual-Luciferase Reporter Assay System (Promega, Germany) and according to the manufacturer’s instruction.
The retrospective study cohort included laryngeal (LSCC) and oropharyngeal squamous cell carcinoma (OPSCC) patients, who were treated at the University Hospital Heidelberg between 1990 and 2008. Paraffin-embedded tissue specimens were provided by the tissue bank of the National Center for Tumor Disease (Institute of Pathology, University Hospital Heidelberg) after approval by the local institutional review board (ethic vote: 206/2005). The study was performed according to the ethical standards of the Declaration of Helsinki. For all tumor samples, clinical and follow-up data were available from the Department of Otolaryngology, Head and Neck Surgery at the University Hospital Heidelberg.
Tissue microarray and IHC staining
TMAs were generated as previously described . Briefly, hematoxylin and eosin stained sections were cut from each donor block and a certified pathologist defined representative tumor regions. Small tissue cylinders with a diameter of 0.6 mm were taken from selected areas of each donor block using a tissue chip micro-arrayer (Beecher Instruments, Silver Spring, MD, USA) and transferred to a recipient paraffin block. The recipient paraffin block was cut in 2 μm paraffin sections using standard techniques. Immunohistochemistry was conducted with an anti-KLK6 antibody (R&D Systems, Germany) listed in Additional file 5: Table S2 using the TSA Amplification Kit (Perkin Elmer, Germany) according to the manufacturer’s instruction. Counterstaining was done with hematoxylin to visualize tissue integrity.
TMAs were scanned using the Nanozoomer HT Scan System (Hamamatsu Photonics, Japan) capable of scanning whole slides. The Nanozoomer HT Scan System generates digital images of full tissue sections, allowing large-scale histologic evaluation across the complete section. The slides were scanned at a 40-fold magnification (0.23 μm/pixel). Three experienced observers analyzed scanned slides without prior knowledge on the expected results using the NDP Viewer software (version 1.1.27). The semi-quantitative analysis was performed according to two independent categories: first, the number of stained tumor cells (ranging from 1 to 4: score 1 = no positive cells, score 2 = less than 33 %, score 3 = between 34 and 66 %, and score 4 = more than 66 % positive cells), and second, the staining intensity (ranging from 1 to 4: score 1 = no staining, score 2 = weak staining, score 3 = moderate staining, and score 4 = high staining). Subsequently, both scores were multiplied resulting in the final expression score ranging from 1–16, and patient subgroups were arranged according to KLK6high or KLK6low expression.
Statistical analysis was done using SPSS (version 21) and SAS (version 9.3) statistics software. Differences between groups were assessed using Chi square test. Overall survival (OS) was calculated as the time from the date of primary tumor diagnosis to the date of cancer-related death within the follow-up interval (events). Survival times of patients who were alive or were dead due to causes other than cancer were censored. Progression-free survival (PFS) was calculated from the date of primary tumor diagnosis to the date of the first local recurrence, lymph node or distant metastasis, second primary carcinoma, or date of cancer-related death within the follow-up period (events). Patients without progression (no event) or cancer-unrelated death were censored. The method of Kaplan-Meier was used to estimate survival distributions. Differences between groups were determined by log-rank tests. A multivariate Cox proportional hazard model was used to assess the association between KLK6 expression scores and overall and progression-free survival of cancer patients from both cohorts, together with the covariates age (continuous variable), clinical stage (IV vs. I–III), gender (female vs. male), TNM, grading, alcohol and tobacco consumption (never vs. former and current consumer). The validity of the proportional hazards assumption was tested with the Supreme Test for proportional hazards assumption and was met for all covariates. In all statistical tests, a p-value of 0.05 or below was considered as statistically significant.
We gratefully acknowledge Leoni Erdinger, Ines Kaden, Nataly Henfling, Antje Schuhmann, Ingeborg Vogt and Melanie Sator-Schmitt for excellent technical assistance, Dana Holzinger for providing data on the HPV status, Sarika Sharma and Pila Bayo for fruitful discussion and critical proofreading of the manuscript. We thank the tissue bank of the National Center for Tumor Disease (Institute of Pathology, University Hospital Heidelberg) for providing tumor specimens of OPSCC patients. This work was supported by the German Research Foundation (HE 5760/3-1 to. J.H.).
- Borgono CA, Diamandis EP. The emerging roles of human tissue kallikreins in cancer. Nat Rev Cancer. 2004;4:876–90.View ArticlePubMedGoogle Scholar
- Sotiropoulou G, Pampalakis G, Diamandis EP. Functional roles of human kallikrein-related peptidases. J Biol Chem. 2009;284:32989–94.View ArticlePubMed CentralPubMedGoogle Scholar
- Bayani J, Diamandis EP. The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6). Clin Chem Lab Med. 2012;50:211–33.View ArticleGoogle Scholar
- Prassas I, Eissa A, Poda G, Diamandis EP. Unleashing the therapeutic potential of human kallikrein-related serine proteases. Nat Rev Drug Discov. 2015;14:183–202.View ArticlePubMedGoogle Scholar
- Oikonomopoulou K, Diamandis EP, Hollenberg MD. Kallikrein-related peptidases: proteolysis and signaling in cancer, the new frontier. Biol Chem. 2010;391:299–310.PubMedGoogle Scholar
- Borgono CA, Michael IP, Diamandis EP. Human tissue kallikreins: physiologic roles and applications in cancer. Mol Cancer Res. 2004;2:257–80.PubMedGoogle Scholar
- Seiz L, Dorn J, Kotzsch M, Walch A, Grebenchtchikov NI, Gkazepis A, et al. Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients. Biol Chem. 2012;393:391–401.View ArticlePubMedGoogle Scholar
- Krenzer S, Peterziel H, Mauch C, Blaber SI, Blaber M, Angel P, et al. Expression and function of the kallikrein-related peptidase 6 in the human melanoma microenvironment. J Invest Dermatol. 2011;131:2281–8.View ArticlePubMed CentralPubMedGoogle Scholar
- Bayani J, Marrano P, Graham C, Zheng Y, Li L, Katsaros D, et al. Genomic instability and copy-number heterogeneity of chromosome 19q, including the kallikrein locus, in ovarian carcinomas. Mol Oncol. 2011;5:48–60.View ArticlePubMed CentralPubMedGoogle Scholar
- Bayani J, Paliouras M, Planque C, Shan SJ, Graham C, Squire JA, et al. Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer. Mol Oncol. 2008;2:250–60.View ArticlePubMedGoogle Scholar
- Paliouras M, Diamandis EP. Coordinated steroid hormone-dependent and independent expression of multiple kallikreins in breast cancer cell lines. Breast Cancer Res Treat. 2007;102:7–18.View ArticlePubMedGoogle Scholar
- Yousef GM, Luo LY, Scherer SW, Sotiropoulou G, Diamandis EP. Molecular characterization of zyme/protease M/neurosin (PRSS9), a hormonally regulated kallikrein-like serine protease. Genomics. 1999;62:251–9.View ArticlePubMedGoogle Scholar
- Olkhov-Mitsel E, Van der Kwast T, Kron KJ, Ozcelik H, Briollais L, Massey C, et al. Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer. Epigenetics. 2012;7:1037–45.View ArticlePubMed CentralPubMedGoogle Scholar
- Pampalakis G, Prosnikli E, Agalioti T, Vlahou A, Zoumpourlis V, Sotiropoulou G. A tumor-protective role for human kallikrein-related peptidase 6 in breast cancer mediated by inhibition of epithelial-to-mesenchymal transition. Cancer Res. 2009;69:3779–87.View ArticlePubMedGoogle Scholar
- Pampalakis G, Sotiropoulou G. Multiple mechanisms underlie the aberrant expression of the human kallikrein 6 gene in breast cancer. Biol Chem. 2006;387:773–82.PubMedGoogle Scholar
- Breitenbach U, Tuckermann JP, Gebhardt C, Richter KH, Furstenberger G, Christofori G, et al. Keratinocyte-specific onset of serine protease BSSP expression in experimental carcinogenesis. J Invest Dermatol. 2001;117:634–40.View ArticlePubMedGoogle Scholar
- Bourcier C, Jacquel A, Hess J, Peyrottes I, Angel P, Hofman P, et al. p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1)-dependent signaling contributes to epithelial skin carcinogenesis. Cancer Res. 2006;66:2700–7.View ArticlePubMedGoogle Scholar
- Ignatenko NA, Babbar N, Mehta D, Casero Jr RA, Gerner EW. Suppression of polyamine catabolism by activated Ki-ras in human colon cancer cells. Mol Carcinog. 2004;39:91–102.View ArticlePubMedGoogle Scholar
- Ignatenko NA, Zhang H, Watts GS, Skovan BA, Stringer DE, Gerner EW. The chemopreventive agent alpha-difluoromethylornithine blocks Ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells. Mol Carcinog. 2004;39:221–33.View ArticlePubMedGoogle Scholar
- Bayani J, Kuzmanov U, Saraon P, Fung WA, Soosaipillai A, Squire JA, et al. Copy number and expression alterations of miRNAs in the ovarian cancer cell line OVCAR-3: impact on kallikrein 6 protein expression. Clin Chem. 2013;59:296–305.View ArticlePubMedGoogle Scholar
- Chow TF, Crow M, Earle T, El-Said H, Diamandis EP, Yousef GM. Kallikreins as microRNA targets: an in silico and experimental-based analysis. Biol Chem. 2008;389:731–8.View ArticlePubMedGoogle Scholar
- Anisowicz A, Sotiropoulou G, Stenman G, Mok SC, Sager R. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med. 1996;2:624–36.PubMed CentralPubMedGoogle Scholar
- White NM, Bui A, Mejia-Guerrero S, Chao J, Soosaipillai A, Youssef Y, et al. Dysregulation of kallikrein-related peptidases in renal cell carcinoma: potential targets of miRNAs. Biol Chem. 2010;391:411–23.View ArticlePubMedGoogle Scholar
- Gabril M, White NM, Moussa M, Chow TF, Metias SM, Fatoohi E, et al. Immunohistochemical analysis of kallikrein-related peptidases in the normal kidney and renal tumors: potential clinical implications. Biol Chem. 2010;391:403–9.View ArticlePubMedGoogle Scholar
- Petraki CD, Gregorakis AK, Vaslamatzis MM, Papanastasiou PA, Yousef GM, Levesque MA, et al. Prognostic implications of the immunohistochemical expression of human kallikreins 5, 6, 10 and 11 in renal cell carcinoma. Tumour Biol. 2006;27:1–7.View ArticlePubMedGoogle Scholar
- Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer. 2011;11:9–22.View ArticlePubMedGoogle Scholar
- Pai SI, Westra WH. Molecular pathology of head and neck cancer: implications for diagnosis, prognosis, and treatment. Annu Rev Pathol. 2009;4:49–70.View ArticlePubMed CentralPubMedGoogle Scholar
- Kostareli E, Holzinger D, Hess J. New concepts for translational head and neck oncology: lessons from HPV-related oropharyngeal squamous cell carcinomas. Front Head Neck Cancer. 2012;2:1–10.Google Scholar
- Gillison ML, Castellsague X, Chaturvedi A, Goodman MT, Snijders P, Tommasino M, et al. Eurogin Roadmap: Comparative epidemiology of HPV infection and associated cancers of the head and neck and cervix. Int J Cancer. 2013;134:497–507.View ArticlePubMedGoogle Scholar
- Schmalhofer O, Brabletz S, Brabletz T. E-cadherin, beta-catenin, and ZEB1 in malignant progression of cancer. Cancer Metastasis Rev. 2009;28:151–66.View ArticlePubMedGoogle Scholar
- Ogawa K, Utsunomiya T, Mimori K, Tanaka F, Inoue H, Nagahara H, et al. Clinical significance of human kallikrein gene 6 messenger RNA expression in colorectal cancer. Clin Cancer Res. 2005;11:2889–93.View ArticlePubMedGoogle Scholar
- Kim JT, Song EY, Chung KS, Kang MA, Kim JW, Kim SJ, et al. Up-regulation and clinical significance of serine protease kallikrein 6 in colon cancer. Cancer. 2011;117:2608–19.View ArticlePubMedGoogle Scholar
- Nagahara H, Mimori K, Utsunomiya T, Barnard GF, Ohira M, Hirakawa K, et al. Clinicopathologic and biological significance of kallikrein 6 overexpression in human gastric cancer. Clin Cancer Res. 2005;11:6800–6.View ArticlePubMedGoogle Scholar
- Liu X, Xiong H, Li J, He Y, Yuan X. Correlation of hK6 expression with tumor recurrence and prognosis in advanced gastric cancer. Diagn Pathol. 2013;8:62.View ArticlePubMed CentralPubMedGoogle Scholar
- Ruckert F, Hennig M, Petraki CD, Wehrum D, Distler M, Denz A, et al. Co-expression of KLK6 and KLK10 as prognostic factors for survival in pancreatic ductal adenocarcinoma. Br J Cancer. 2008;99:1484–92.View ArticlePubMed CentralPubMedGoogle Scholar
- White NM, Mathews M, Yousef GM, Prizada A, Popadiuk C, Dore JJ. KLK6 and KLK13 predict tumor recurrence in epithelial ovarian carcinoma. Br J Cancer. 2009;101:1107–13.View ArticlePubMed CentralPubMedGoogle Scholar
- Kountourakis P, Psyrri A, Scorilas A, Camp R, Markakis S, Kowalski D, et al. Prognostic value of kallikrein-related peptidase 6 protein expression levels in advanced ovarian cancer evaluated by automated quantitative analysis (AQUA). Cancer Sci. 2008;99:2224–9.View ArticlePubMedGoogle Scholar
- Nathalie HV, Chris P, Serge G, Catherine C, Benjamin B, Claire B, et al. High kallikrein-related peptidase 6 in non-small cell lung cancer cells: an indicator of tumour proliferation and poor prognosis. J Cell Mol Med. 2009;13:4014–22.View ArticlePubMedGoogle Scholar
- Talieri M, Zoma M, Devetzi M, Scorilas A, Ardavanis A. Kallikrein-related peptidase 6 (KLK6) gene expression in intracranial tumors. Tumour Biol. 2012;33:1375–83.View ArticlePubMedGoogle Scholar
- Drucker KL, Paulsen AR, Giannini C, Decker PA, Blaber SI, Blaber M, et al. Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma. Neuro Oncol. 2013;15:305–18.View ArticlePubMed CentralPubMedGoogle Scholar
- Patsis C, Yiotakis I, Scorilas A. Diagnostic and prognostic significance of human kallikrein 11 (KLK11) mRNA expression levels in patients with laryngeal cancer. Clin Biochem. 2012;45:623–30.View ArticlePubMedGoogle Scholar
- Foteinou E, Kontos CK, Giotakis AI, Scorilas A. Low mRNA expression levels of kallikrein-related peptidase 4 (KLK4) predict short-term relapse in patients with laryngeal squamous cell carcinoma. Biol Chem. 2014;395:1051–62.View ArticlePubMedGoogle Scholar
- Yamano Y, Uzawa K, Shinozuka K, Fushimi K, Ishigami T, Nomura H, et al. Hyaluronan-mediated motility: a target in oral squamous cell carcinoma. Int J Oncol. 2008;32:1001–9.PubMedGoogle Scholar
- Ishige S, Kasamatsu A, Ogoshi K, Saito Y, Usukura K, Yokoe H, et al. Decreased expression of kallikrein-related peptidase 13: possible contribution to metastasis of human oral cancer. Mol Carcinog. 2014;53:557–65.View ArticlePubMedGoogle Scholar
- Klucky B, Mueller R, Vogt I, Teurich S, Hartenstein B, Breuhahn K, et al. Kallikrein 6 induces e-cadherin shedding and promotes cell proliferation, migration, and invasion. Cancer Res. 2007;67:8198–206.View ArticlePubMedGoogle Scholar
- Henkhaus RS, Gerner EW, Ignatenko NA. Kallikrein 6 is a mediator of K-RAS-dependent migration of colon carcinoma cells. Biol Chem. 2008;389:757–64.View ArticlePubMed CentralPubMedGoogle Scholar
- Pampalakis G, Arampatzidou M, Amoutzias G, Kossida S, Sotiropoulou G. Identification and analysis of mammalian KLK6 orthologue genes for prediction of physiological substrates. Comput Biol Chem. 2008;32:111–21.View ArticlePubMedGoogle Scholar
- Ravindran G, Sawant SS, Hague A, Kingsley K, Devaraj H: Association of differential beta-catenin expression with Oct-4 and Nanog in oral squamous cell carcinoma and their correlation with clinicopathological factors and prognosis. Head Neck 2014. doi: 10.1002/hed.23699. [Epub ahead of print].Google Scholar
- Warrier S, Bhuvanalakshmi G, Arfuso F, Rajan G, Millward M, Dharmarajan A. Cancer stem-like cells from head and neck cancers are chemosensitized by the Wnt antagonist, sFRP4, by inducing apoptosis, decreasing stemness, drug resistance and epithelial to mesenchymal transition. Cancer Gene Ther. 2014;21:381–8.View ArticlePubMedGoogle Scholar
- Sun S, Liu S, Duan SZ, Zhang L, Zhou H, Hu Y, et al. Targeting the c-Met/FZD8 signaling axis eliminates patient-derived cancer stem-like cells in head and neck squamous carcinomas. Cancer Res. 2014;74:7546–59.View ArticlePubMedGoogle Scholar
- Michel N, Heuze-Vourc’h N, Lavergne E, Parent C, Jourdan ML, Vallet A, et al. 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. Biol Chem. 2014;395:1015–25.View ArticlePubMedGoogle Scholar
- Niyazi M, Niyazi I, Belka C. Counting colonies of clonogenic assays by using densitometric software. Radiat Oncol. 2007;2:4.View ArticlePubMed CentralPubMedGoogle Scholar
- Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315–9.View ArticlePubMedGoogle Scholar
- Leibold JS, Riehl A, Hettinger J, Durben M, Hess J, Angel P: Keratinocyte-specific deletion of the receptor RAGE modulates the kinetics of skin inflammation in vivo. J Invest Dermatol 2013;133:2400–6.Google Scholar
- Roesch Ely M, Nees M, Karsai S, Magele I, Bogumil R, Vorderwulbecke S, et al. Transcript and proteome analysis reveals reduced expression of calgranulins in head and neck squamous cell carcinoma. Eur J Cell Biol. 2005;84:431–44.View ArticlePubMedGoogle Scholar
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