Alternative splicing variant of kallikrein-related peptidase 8 as an independent predictor of unfavorable prognosis in lung cancer.
KLK8 was originally cloned from a human skin library (9) as a homolog of a gene encoding mouse neuropsin. The KLK8 peptidase is present in numerous human tissues (10) and is involved in several physiological and pathologic processes (11, 12). Abnormal KLK8 transcripts and/or the KLK8 protein have been found in several malignancies, including uterine endometrial carcinoma and ovarian, lung, and neck cancers (11, 13-15). Five alternative mRNA variants encoded by KLK8, including the regular form, have been described (7). Type 1 and type 2 KLK8 mRNA variants (KLK8-T1 and KLK8-T2) produce 2 zymogens that differ only in their propeptide sequences. In this case, alternative splicing produces the same final active protein, but the 2 zymogens are released in a cell type-dependent manner and are activated differently (16). The KLK8-T3 mRNA variant encodes a truncated form of the KLK8 protein. The KLK8-T4 variant lacks exons 3-5. It encodes a putative protein of 32 amino acid residues that contains the KLK8 signal peptide and another peptide that is not related to KLK8 (17). The KLK8-T3 and KLK8-T4 mRNAs are abundant in many tissues (brain, pancreas, skin) and are overproduced in ovarian cancers (17).
We have examined the pattern ofKLK8 mRNAs in NSCLC samples by reverse-transcription PCR (RTPCR) and DNA sequencing. We identified 6 alternatively spliced transcripts, of which KLK8-T3 and KLK8-T4 were the most abundant. The concentrations of these 2 splice variants in a cohort of NSCLC patients were then measured by quantitative real-time PCR. Finally, we found that a splice variant mRNA, KLK8-T4, may be an independent indicator of a poor prognosis for lung cancer patients.
Materials and Methods
Matched samples of tumor and nontumor tissue were obtained from 60 patients who had undergone lung cancer resection as their primary therapy without preoperative radiation or chemotherapy. Tumor and nontumor tissue samples were selected by a pathologist from each fresh surgical sample, immediately frozen in liquid nitrogen, and stored at -80 [degrees]C. The nonmalignant tissue samples were taken from sites at least 3 cm away from the edge of the tumor. Histologic diagnosis was performed, and tumor grade was determined in accordance with the WHO classification of lung tumors. The tissue samples were banked with informed consent, in compliance with the Helsinki Accord and French bioethical regulations. The methods used for RNA extraction, cDNA synthesis, and conventional RT-PCR are described in the Supplemental Data section of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/ content/vol56/issue6.
QUANTITATIVE REAL-TIME RT-PCR ANALYSIS
To analyze KLK8 gene expression, we based the design and synthesis of primer sets on the published mRNA sequence of KLK8-T1 (set KLK8, GenBank accession no. NM_007196), KLK8-T3 (NM_144506), and KLK8-T4 (NM_144507) (Table 1). Primers were designed to target 2 exons. The amounts of 18S rRNA and mRNAs encoded by KLK5, KLK6, KLK7, KLK10,
KLK11, KLK13, and KLK14 were measured as previously described (18, 19) (see Supplemental Data section in the online Data Supplement). Real-time PCR was carried out in iCycler 96-well PCR plates on an iCycler iQ Real-Time PCR System (Bio-Rad Laboratories) with the SYBR Green I chemistry (see Supplemental Data section in the online Data Supplement). Each assay included 2 no-template controls, cDNA samples in duplicate, and serial dilutions of the appropriate plasmid DNA calibrator for constructing a calibration curve. The concentrations of all samples were calculated by plotting their quantification cycles against the calibration curve. The amount of the target molecule was then normalized by dividing by the amount of the endogenous reference (18S rRNA). Values are expressed in arbitrary units.
The KLK8 mRNAs were used to classify KLK8 gene activity as negative or positive. The [chi square] test or the Fisher exact test was used as appropriate to analyze associations between KLK8 gene activity and other qualitative variables.
We used the Kaplan-Meier method for constructing overall survival (OS) curves to demonstrate differences in the survival of KLK8 mRNA-positive and KLK8 mRNA-negative patients. The OS time was defined as the time between the initial surgery and death. The survival times of patients who were still alive were noted along with the dates of the last follow-up appointment. The log-rank test was used to determine statistically significant differences between OS curves. The impact of KLK8 gene activity on patient OS was assessed as the relative risk (RR) of death in the group with high KLK8 activity, as calculated with the Cox univariate and multivariate proportional hazard regression models. In the multivariate analysis, we adjusted for the clinical and pathologic variables that could affect survival, including sex, age, stage of disease, histologic type, residual tumor size, and rate of smoking.
IDENTIFICATION OF KLK8 mRNA VARIANTS IN LUNG TISSUE
To carry out the RT-PCR, we used RNA prepared from nonpathologic and tumoral lung tissues and the specific primer set to amplify the entire coding sequence of KLK8 mRNA. The RT-PCR produced several products (Fig. 1), and we determined the nucleotide sequences of 6 of them. The sequences of 5 products were identical to those of the regular KLK8 mRNA (9) and alternative KLK8 gene transcript types 2-5 (11, 17).We identified 1 new alternatively spliced mRNA, KLK8 type 6 (KLK8-T6), which lacked exon 4. For this variant, the alternative splicing creates a stop codon that prematurely terminates translation at amino acid residue 80. The predicted KLK8-T6 protein has the signal peptide, which is necessary for the secretion of the type 1 and type 2 forms (16), suggesting that the type 6 variant is secreted. The truncated protein has only 1 amino acid residue of the catalytic triad and probably has no serine protease activity.
All 6 KLK8 mRNA forms were found mainly in lung cancer tissues; little or none was found in nonpathologic lung tissue (Fig. 1). The KLK8-T3 and KLK8-T4 mRNA variants appeared to be the most abundant forms in lung cancer.
REAL-TIME QUANTIFICATION OF mRNA KLK8 ISOFORMS IN NSCLC
We assayed the activity of the KLK8 gene in lung tumors and nonpathologic lung tissues with 3 primer sets designed for quantitative real-time PCR analysis (Table 1). The first set (total KLK8 mRNA) was used to measure all KLK8 gene transcripts except KLK8-T4; the other 2 sets were used to quantify KLK8-T3 or KLK8-T4 mRNA. We evaluated the specificity of PCR reactions by running quantitative real-time RT-PCR experiments with the primers specific for KLK8-T3 or KLK8-T4 in samples containing cloned cDNA that corresponded to the other transcript. Each transcript was amplified only by its specific primer pairs; the degree of cross-reaction was negligible. The dynamic ranges for KLK8-T3 and KLK8-T4 were 10-106 copies. The Pearson correlation coefficient for the calibration curves was 0.99. The analytical limit of quantification was 15 copies per reaction (see Supplemental Data section in the online Data Supplement).
The total concentration of KLK8 mRNA in 60
NSCLC samples was significantly higher (P < 0.0001) than that in the paired, apparently unaffected control tissues (Fig. 2). Similarly, KLK8-T3 and KLK-T4 mRNAs were more abundant in NSCLC samples than in paired nonpathologic tissue samples (P < 0.0001).
We compared the mRNA amounts for total KLK8, KLK8-T3, and KLK8-T4 in patients who been classified according to conventional clinicopathologic parameters (Table 2). We used [chi square] analysis to identify an optimal cutoff value for each variable on the basis of the ability of the variable to predict the OS time of the study population. With this cutoff, we separated the lung tumors into positive and negative groups (i.e., above and below the cutoff) and were able to correlate patients with KLK8 mRNA-positive and KLK8-T3-positive tumors with the squamous cell carcinoma histotype (P < 0.05). The KLK8-T4-positive tumors were mainly of T3-T4 status, although the result was marginally nonsignificant (P = 0.066). We found no relationship between KLK8, KLK8-T3, or KLK8-T4 mRNA status and tumor grade, size, nodal status, or stage of cancer.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
KLK8 mRNA ISOFORMS AND OS
We examined several variables, including total KLK8 mRNA and the KLK8-T3 and KLK8-T4 mRNAs. The KLK8 mRNAs were considered alone or as a ratio with the KLK5, KLK6, KLK7, KLK10, KLK11, KLK13, or KLK14 mRNA in the same samples. An optimal cutoff value was identified for each variable by x2 analysis, as described above, and the lung tumors were stratified into positive and negative groups (above and below the cutoff). Table 3 shows the strength of the association between positive tumors and OS. Univariate analysis indicated that clinical stage was significantly associated with a poor prognosis (RR = 4.14; P = 0.001), as was the presence of KLK8, KLK8-T3, or KLK8-T4 mRNA [RR = 2.56 (P = 0.043), 2.64 (P =0.037), and 3.37 (P = 0.009), respectively]. Several ratios also were significantly associated with OS (Table 3). The KLK8, KLK8T3, and KLK8-T4 mRNA ratios with the clearest associations with OS were KLK8/KLK13 (RR = 6.32; P = 0.003), KLK8-T3/KLK13 (RR = 4.25; P = 0.009), and KLK8-T4/KLK11 (RR = 8.16; P = 0.001). Multivariate Cox regression analysis did not confirm the relationship between variables that included KLK8-T3 mRNA and OS. Conversely, the KLK8/KLK10, KLK8/KLK11, and KLK8/KLK13 mRNA ratios remained independent prognostic factors for survival. Almost all of the variables that included KLK8-T4 mRNA were significantly associated with OS in the Cox multivariate regression analysis (Table 3). The RR of cancer-related death was >8-fold higher (RR = 8.37; P = 0.001) for patients with a KLK8-T4/KLK11 mRNA ratio above the cutoff than for those with a KLK8-T4/KLK11 ratio below the cutoff. Kaplan-Meier survival curves further demonstrated that patients with KLK8-T4/KLK11 positive tumors had substantially shorter OS times (P < 0.001) than those with KLK8-T4/KLK11-negative tumors (Fig. 3).
The KLK8 gene encodes at least 6 mRNA variants in lung tissue, including the classic KLK8-T1 form. The alternative splicing events imply exon skipping (mRNAs KLK8-T3, -T4, -T5, and -T6) and exon extension (KLK8-T2), which are the most common mechanisms generating mRNA variants in the KLK gene family (7, 8). The KLK8 gene is much more active in NSCLC (P < 0.0001) than in healthy lung tissue, as it is for several other malignancies, including uterine endometrial carcinoma and ovarian and neck cancers (13-15). Several KLK8 mRNAs are abnormally abundant in NSCLC, suggesting that a common mechanism, such as an increase in the transcription rate of the KLK8 gene, affects the steady-state concentration of these mRNAs; however, the concentrations of each splice variant are not similarly increased. Therefore, specific mechanism(s) may also regulate the steady-state concentration of individual KLK8 splice variants. Traditional models of how alternative splicing is regulated involve auxiliary splicing factors that bind to the premRNA and enhance or repress the ability of the spliceosome to recognize particular splice sites (20). According to these models, changes in the relative distributions of auxiliary factors affect the pattern of alternative splicing. Various auxiliary splicing factors are up-regulated in lung cancer (21), and differential production of alternative splice variants seems to be common in this disease (22). We therefore postulate that the differential production of alternative KLK8 mRNAs in NSCLC is due to overall alterations in the splicing machinery.
Our findings concerning the KLK8 mRNA variable are in general agreement with a previous study on the expression of the KLK8 gene in lung cancer (11). In both of these studies, the primer set used to evaluate KLK8 gene activity hybridizes with all of the alternative KLK8 gene transcripts, except KLK8-T4 (Table 1). We found no statistically significant associations between KLK8 gene activity and several clinicopathologic variables (grade, stage, size or status of the tumor, and nodal status) except for the squamous cell carcinoma histotype (P = 0.018). Neither Sher et al. (11) nor we found any association between KLK8 activity and OS by multivariate analysis [RR = 1.45 (P = 0.486) in our Cox multivariate analysis]. Sher et al., however, reported that early-stage (I-II) NSCLC patients with high KLK8 gene expression in their tumors had significantly longer remission times and lower rates of recurrence. Similar observations were reported in patients with ovarian cancer (17), with a multivariate analysis showing high KLK8 mRNA production to be associated with disease-free survival but not with OS. The duration of progression-free survival in cancer patients depends on the probability for and percentages of tumor cells to pass from one step of the metastatic process to the next. These steps include local invasion, intravasation of cells from the primary tumor into the circulatory system, survival of these cells within the blood or lymphatic system, evasion of the immune system, arrest at a secondary site distant from the site of origin, extravasation, initiation of either intra- or extravascular growth within this secondary site, and, finally, maintenance of growth leading to the formation of overt, vascularized, clinically detectable metastases (23). Overproduction of type 1 and type 2 KLK8 mRNAs has been shown to decrease the in vitro invasiveness of lung cancer cells (11). These protective effects of KLK8-T1 and KLK8-T2 mRNAs have been ascribed to the proteolysis of extracellular fibronectin by the encoded KLK8 peptidase, KLK8-mediated degradation of fibronectin-suppressed integrin signaling, and decreased lung cancer cell motility through inhibition of actin polymerization. In nude mice, production of these splice variants decreases tumor growth and reduces intravasation (11). The protective role of KLK8 in invasiveness may explain the longer disease-free survival times of ovarian cancer patients with high concentrations of KLK8 mRNA in their cancer tissue (17, 24), or higher concentrations of the KLK8 peptidase in their tissues (13, 25) or ascites fluids (26). Collectively, these findings suggest that the KLK8 peptidase may influence the initial course of ovarian and lung cancers by delaying some early steps of the metastatic process without having any implication on their final outcomes.
[FIGURE 3 OMITTED]
In contrast, our multivariate Cox proportional hazards regression analysis and the Kaplan-Meier survival curves indicate that KLK8-T4 mRNA is an independent predictor of an unfavorable prognosis in lung cancer. Although several studies have shown that splice variants of various KLK genes are differentially regulated in cancer tissues (8), our results are the first evidence of the clinical value of one alternative transcript of a KLK gene. Cancer deaths are generally due to the physiological effects of local or distant metastases, rather than to the primary tumor. KLK8-T4 mRNA production may therefore have a negative impact on the final NSCLC outcome by facilitating the occurrence of life-threatening metastases. Our observations, together with those of Sher et al. on KLK8 (11), suggest that the KLK8 gene generates 2 products that have opposite actions on NSCLC progression and metastasis. Divergent biological functions of splice variants have previously been reported for 2 isoforms encoded by ERBB4 [v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian)] that exhibit markedly opposing effects on mammary epithelium growth and differentiation (27). Moreover, metastasis requires functionally distinct classes ofgenes and activities that regulate metastasis initiation, progression, and virulence functions (23). In this context, it is conceivable that the KLK8-T1 and KLK8-T4 mRNAs participate in distinct metastatic processes. Given that KLK8 limits the early steps of metastasis, the prometastatic activity of the KLK8-T4 mRNA could be achieved through actions on later steps, such as the initial seeding or persistent growth of metastatic tumor cells in the target site. For example, expression of the hypoxia-regulated gene LOX (lysyl oxidase) predicts relapse in human breast tumors (28), but recent studies suggest that systemic secretion of lysyl oxidase into the lung and liver might facilitate the homing of disseminated cancer cells to these organs through the formation of a prometastatic microenvironment (29).
KLK8 has recently been reported to be downregulated in nodal metastasis, compared with primary head and neck squamous cell carcinoma (15). The existence of a similar situation in lung metastases would imply actions of the KLK8-T4 mRNA at the level of the primary tumor site that have long-term consequences. Long-term effects have been described in antitumoral immunity. For example, distinct antitumoral immune profiles involving effector memory T cells are generated at the primary tumor site in colorectal cancer. Because of their memory properties, effector memory T cells may provide long-term protection against outgrowth of disseminated occult tumor cells after surgical resection of primary tumors (30). This long-term action might explain why the amplitude of the adaptive immune reaction within the primary tumor was found to be a better predictor of survival in colon cancer than traditional clinical parameters (31). Patients with cancers at nonmetastatic stages had prognoses as poor as patients with metastatic tumors if they presented a low intratumoral adaptive immune reaction. Conversely, patients with metastatic tumors eliciting a high intratumoral immune reaction had a better prognosis. Little is known about the influence of the products of the KLK family on the adaptive immune reaction. Immunoregulatory functions of KLK3 have been described. Kennedy-Smith et al. (32) showed that KLK3 inhibited mitogen and recall antigen-induced T-cell proliferation. Aalamian et al. (33) demonstrated KLK3 inhibition of dendritic cell maturation. Clearly, additional experiments are necessary to elucidate the exact mechanism by which the KLK8-T4 mRNA influences the OS of patients with NSCLC and to determine whether a balance between the KLK8 peptidase and KLK8-T4 mRNA or protein is involved in NSCLC progression.
The KLK8-T4/KLK11 mRNA ratio in lung cancer tissues appears to be a much better predictor of OS (RR = 8.37; P = 0.001) than KLK8-T4 mRNA alone (RR = 3.90; P = 0.016) or pathologic TNM staging (RR = 3.63; P = 0.003). Our previous study found no correlation between KLK11 gene expression and OS in NSCLC (19). Two other variables (KLK7, KLK10) that are not linked to OS in lung cancer (18, 19) have a similar effect on the prognostic value of KLK8-T4 mRNA. One possible explanation is that the basal transcription rate of several KLK genes (KLK7, KLK8, KLK10, KLK11) is concomitantly altered in some individuals because of polymorphisms in common regulatory factors. In these patients, the KLK8-T4 mRNA concentration would not necessarily be related to cancer aggressiveness but would reflect the individual variation in the KLK8 basal transcription rate. Thus, the other variables (i.e., KLK7, KLK10, KLK11) in the 2-gene index would operate as internal controls to normalize the KLK8-T4 mRNA values for changes in the basal transcription rate unrelated to the pathologic status, thereby improving the prognostic significance of the KLK8-T4 variable. As an example, the expression of multiple KLK genes is coordinated in breast cancer cell lines (34). Further studies are required to determine whether the basal expression ofKLK genes in lung tissue is governed by a locus control region, as in other clustered gene families, or whether individual genes are coregulated by the same regulatory factors. Alternatively, these findings could indicate that several KLKs cooperate with KLK8-T4 mRNA production for the occurrence of life-threatening metastases in NSCLC. Several studies found KLKs to be involved in cancer progression through actions on tumor cell growth, invasion, and angiogenesis (35). For example, KLK11 may play a role in breast cancer progression by increasing the bioavailability of insulinlike growth factors via degradation of insulinlike growth factor-binding protein 3 (36). KLK5, KLK13, and KLK14 may also contribute to tumor cell invasion via degradation of extracellular matrix components (37-39). Finally, several KLKs regulate proteinase-activated receptor-mediated signaling through receptor activation or disarming. These G protein-coupled receptors may play roles in cancer-associated inflammation and can promote tumor growth and invasion (40).
In summary, we have obtained evidence that an alternative transcript of the KLK8 gene is an independent predictor of an unfavorable prognosis in NSCLC. The KLK8-T4 /KLK11 mRNA index is a better predictor of OS than clinical stage or the concentration of KLK8-T4 mRNA alone. This 2-gene index may provide a new prognostic marker for NSCLC.
Acknowledgments: We thank Dr. Bruno Giraudeau for helpful suggestions and Dr. Sandra Regina for clinical information. The English text was edited by Dr. Owen Parkes.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: The authors benefited from contributions from INSERM, Ligue Contre le Cancer Region Centre, bioMerieux, and Association pour la Recherche sur le Cancer (grant 7935). C. Planque benefited from a fellowship from the Region Centre and INSERM. Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
(1.) Rami-Porta R, Crowley JJ, Goldstraw P. The revised TNM staging system for lung cancer. Ann Thorac Cardiovasc Surg 2009;15:4-9.
(2.) Singhal S, Miller D, Ramalingam S, Sun SY. Gene expression profiling of non-small cell lung cancer. Lung Cancer 2008;60:313-24.
(3.) Bianchi F, Nuciforo P, Vecchi M, Bernard L, Tizzoni L, Marchetti A, et al. Survival prediction of stage I lung adenocarcinomas by expression of 10 genes. J Clin Invest 2007;117:3436-44.
(4.) RazDJ, Ray MR, Kim JY, He B,Taron M, Skrzypski M, et al. A multigene assay is prognostic of survival in patients with early-stage lung adenocarcinoma. Clin Cancer Res 2008;14:5565-70.
(5.) Grosso AR, Martins S, Carmo-Fonseca M. The emerging role of splicing factors in cancer. EMBO Rep 2008;9:1087-93.
(6.) Emami N, Diamandis EP. Utility of kallikrein-related peptidases (KLKs) as cancer biomarkers. Clin Chem 2008;54:1600-7.
(7.) Kurlender L, Borgono C, Michael IP, Obiezu C, Elliott MB, Yousef GM, Diamandis EP. A survey of alternative transcripts of human tissue kallikrein genes. Biochim Biophys Acta 2005;1755:1-14.
(8.) Tan OL, Whitbread AK, Clements JA, Dong Y. Kallikrein-related peptidase (KLK) family mRNA variants and protein isoforms in hormone-related cancers: Do they have a function? Biol Chem 2006;387:697-705.
(9.) Yoshida S, Taniguchi M, Hirata A, Shiosaka S. Sequence analysis and expression of human neuropsin cDNA and gene. Gene 1998;213:9-16.
(10.) Shaw JL, Diamandis EP. Distribution of 15 human kallikreins in tissues and biological fluids. Clin Chem 2007;53:1423-32.
(11.) Sher YP, Chou CC, Chou RH, Wu HM, Chang WS, Chen CH, et al. Human kallikrein 8 protease confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness. Cancer Res 2006;66:11763-70.
(12.) Komatsu N, Saijoh K, Kuk C, Shirasaki F, Takehara K, Diamandis EP. Aberrant human tissue kallikrein levels in the stratum corneum and se rum of patients with psoriasis: dependence on phenotype, severity and therapy. Br J Dermatol 2007;156:875-83.
(13.) Shigemasa K, Tian X, Gu L, Tanimoto H, Underwood LJ, O'Brien TJ, Ohama K. Human kallikrein 8 (hK8/TADG-14) expression is associated with an early clinical stage and favorable prognosis in ovarian cancer. Oncol Rep 2004;11:1153-9.
(14.) Jin H, Nagai N, Shigemasa K, Gu L, Tanimoto H, Yunokawa M, et al. Expression of tumor associated differentially expressed Gene-14 (TADG-14/KLK8) and its protein hK8 in uterine endometria and endometrial carcinomas. Tumour Biol 2006;27:274-82.
(15.) Liu CJ, Liu TY, Kuo LT, Cheng HW, Chu TH, Chang KW, Lin SC. Differential gene expression signature between primary and metastatic head and neck squamous cell carcinoma. J Pathol 2008; 214:489-97.
(16.) Lu ZX, Huang Q, Su B. Functional characterization of the human-specific (type II) form of kallikrein 8, a gene involved in learning and memory. Cell Res 2009;6:259-67.
(17.) Magklara A, Scorilas A, Katsaros D, Massobrio M, Yousef GM, Fracchioli S, et al. The human KLK8 (neuropsin/ovasin) gene: identification of two novel splice variants and its prognostic value in ovarian cancer. Clin Cancer Res 2001;7:806-11.
(18.) Planque C, de Monte M, Guyetant S, Rollin J, Desmazes C, Panel V, et al. KLK5 and KLK7, two members of the human tissue kallikrein family, are differentially expressed in lung cancer. Biochem Biophys Res Commun 2005;329:1260-6.
(19.) Planque C, Ainciburu M, Heuze-Vourc'h N, Regina S, de Monte M, Courty Y. Expression of the human kallikrein genes 10 (KLK10) and 11 (KLK11) in cancerous and non-cancerous lung tissues. Biol Chem 2006;387:783-8.
(20.) Lin S, Fu XD. SR proteins and related factors in alternative splicing. Adv Exp Med Biol 2007;623: 107-22.
(21.) Karni R, de Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol 2007;14:185-93.
(22.) Xi L, Feber A, Gupta V, Wu M, Bergemann AD, Landreneau RJ, et al. Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer. Nucleic Acids Res 2008;36:6535-47.
(23.) Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 2009;9:274-84.
(24.) Borgono CA, Kishi T, Scorilas A, Harbeck N, Dorn J, Schmalfeldt B, et al. Human kallikrein 8 protein is a favorable prognostic marker in ovarian cancer. Clin Cancer Res 2006;12:1487-93.
(25.) Kountourakis P, Psyrri A, Scorilas A, Markakis S, Kowalski D, Camp RL, et al. Expression and prognostic significance of kallikrein-related peptidase 8 protein levels in advanced ovarian cancer by using automated quantitative analysis. Thromb Haemost 2009;101:541-6.
(26.) Kishi T, Grass L, Soosaipillai A, Scorilas A, Harbeck N, Schmalfeldt B, et al. Human kallikrein 8, a novel biomarker for ovarian carcinoma. Cancer Res 2003;63:2771-4.
(27.) Muraoka-Cook RS, Sandahl MA, Strunk KE, Miraglia LC, Husted C, Hunter DM, et al. ErbB4 splice variants Cyt1 and Cyt2 differ by 16 amino acids and exert opposing effects on the mammary epithelium in vivo. Mol Cell Biol 2009;29:493-548.
(28.) Erler JT, Bennewith KL, Nicolau M, Dornhofer N, Kong C, Le QT, et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 2006;440: 1222- 6.
(29.) Erler JT, Bennewith KL, Cox TR, Lang G, Bird D, Koong A, et al. Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell 2009;15:35-44.
(30.) Camus M, Tosolini M, Mlecnik B, Pages F, Kirilovsky A, Berger A, et al. Coordination of intratumoral immune reaction and human colorectal cancer recurrence. Cancer Res 2009;69:2685-93.
(31.) Galon J, Fridman WH, Pages F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res 2007;67: 1883-6.
(32.) Kennedy-Smith AG, McKenzie JL, Owen MC, Davidson PJ, Vuckovic S, Hart DN. Prostate specific antigen inhibits immune responses in vitro: a potential role in prostate cancer. J Urol 2002;168: 741-7.
(33.) Aalamian M, Tourkova IL, Chatta GS, Lilja H, Huland E, Huland H, et al. Inhibition of dendropoiesis by tumor derived and purified prostate specific antigen. J Urol 2003;170:2026-30.
(34.) 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.
(35.) Pampalakis G, Sotiropoulou G Tissue kallikrein proteolytic cascade pathways in normal physiology and cancer. Biochim Biophys Acta 2007; 1776:22-31.
(36.) Sano A, Sangai T, Maeda H, Nakamura M, Hasebe T, Ochiai A. Kallikrein 11 expressed in human breast cancer cells releases insulin-like growth factor through degradation of IGFBP-3. Int J Oncol 2007;30:1493-8.
(37.) Michael IP, Sotiropoulou G, Pampalakis G, Magklara A, Ghosh M, Wasney G, Diamandis EP. Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression. J Biol Chem 2005;280:14628-35.
(38.) Kapadia C, Ghosh MC, Grass L, Diamandis EP. Human kallikrein 13 involvement in extracellular matrix degradation. Biochem Biophys Res Commun 2004;323:1084-90.
(39.) Rajapakse S, Takahashi T. Expression and enzymatic characterization of recombinant human kallikrein 14. Zoolog Sci 2007;24:774-80.
(40.) Hollenberg MD, Oikonomopoulou K, Hansen KK, Saifeddine M, Ramachandran R, Diamandis EP. Kallikreins and proteinase-mediated signaling: proteinase-activated receptors (PARs) and the pathophysiology of inflammatory diseases and cancer. Biol Chem 2008;389:643-51.
Chris Planque, [1,2] Yun-Hee Choi,  Serge Guyetant, [1,2,4] Nathalie Heuze-Vourc'h, [1,2] Laurent Briollais,  and Yves Courty [1,2] *
 University Francois-Rabelais de Tours, Tours, France;  INSERM U  "Proteases et Vectorisation Pulmonaires," Tours, France;  Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada;  Department of Pathology, CHRU de Tours, Tours, France.
* Address correspondence to this author at: INSERM U618, Facultede Medecine, 10 Blvd. Tonnelle, F-37032 Tours Cedex 1, France. Fax +33-2-47-36-60-46; e-mail email@example.com.
Received October 19, 2009; accepted March 8, 2010.
Previously published online at DOI: 10.1373/clinchem.2009.138917
 Nonstandard abbreviations: NSCLC, non-small-cell lung cancer; TNM, tumor-node-metastasis; KLK, kallikrein-related peptidase; KLK8-T1, KLK8 mRNA splice variant type 1; RT-PCR, reverse-transcription PCR; OS, overall survival; RR, relative risk.
 Human genes: KLK8, kallikrein-related peptidase 8; KLK3, kallikrein-related peptidase 3; KLK5, kallikrein-related peptidase 5; KLK6, kallikrein-related peptidase 6; KLK7, kallikrein-related peptidase 7; KLK10, kallikrein-related peptidase 10; KLK11, kallikrein-related peptidase 11; KLK13, kallikrein-related peptidase 13; KLK14, kallikrein-related peptidase 14; ERBB4, v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian); LOX, lysyl oxidase.
Table 1. Characteristics of the primers used for the quantification of the KLK8 transcripts. Primer set Primer sequence KLK8 Fwd: (a) 5'-CCAGAAGAAGTGTGAGGATG-3' Rev: 5'-GGTATAGACGCCAGGTTTG-3' KLK8-T3 Fwd: 5'-GGAGCCTGGGCAGAGAAT-3' Rev: 5'-CCTCCAGAATCGCCCT-3' KLK8-T4 Fwd: 5'-TGGGCAGGGCGATTCT-3' Rev: 5'-CAGTCCAGGTAGCGGCAG-3' Primer set Position Product length KLK8 Exon 5 190 bp Exon 6 KLK8-T3 Exon 2-exon 5 junction 161 bp Exon 6 KLK8-T4 Exon 2-exon 6 junction 132 bp Exon 6 Primer set Detected transcript KLK8 KLK8-T1, -T2, -T3, -T5, and -T6 KLK8-T3 KLK8-T3 KLK8-T4 KLK8-T4 (a) Fwd, forward; Rev, reverse. Table 2. Relationship of KLK8, KLK8-T3, and KLK8-T4 mRNA status with clinicopathologic variables. (a) KLK8 Negative, Positive, Variable n (%) n (%) P Histotype ADC (b) (n = 33) 16 (48.5) 17 (51.5) 0.029 (c) SCC (n = 17) 2 (11.8) 15 (88.2) Other subtypes (n = 10) 5 (50.0) 5 (50.0) Tumor grade Poorly diff (n = 19) 7 (36.8) 12 (63.2) 1.000 (d) Diff (n = 39) 14 (35.9) 25 (64.1) Unknown (n = 2) Tumor size [less than or equal to] 3cm(n = 20) 8 (40.0) 12 (60.0) 1.000 (d) >3cm(n= 40) 15 (37.5) 25 (62.5) Nodal status N0 (n = 40) 15 (37.5) 25 (62.5) 1.000 (d) N1/N2 (n = 20) 8 (40.0) 12 (60.0) Tumor status T1/T2 (n = 47) 20 (42.6) 27 (57.4) 0.334 (d) T3/T4 (n = 13) 3 (23.1) 10 (76.9) Stage I/II (n = 37) 16 (43.2) 21 (56.8) 0.416 (d) III/IV (n = 23) 7 (30.4) 16 (69.6) KLK8-T3 Negative, Positive, Variable n (%) n (%) P Histotype ADC (b) (n = 33) 17 (51.5) 16 (48.5) 0.035 (c) SCC (n = 17) 3 (17.6) 14 (82.4) Other subtypes (n = 10) 6 (60.0) 4 (40.0) Tumor grade Poorly diff (n = 19) 8 (42.1) 11 (57.9) 1.000 (d) Diff (n = 39) 16 (41.0) 23 (59.0) Unknown (n = 2) Tumor size [less than or equal to] 3cm(n = 20) 9 (45.0) 11 (55.0) 1.000 (d) >3cm(n= 40) 17 (42.5) 23 (57.5) Nodal status N0 (n = 40) 18 (45.0) 22 (55.0) 0.787 (d) N1/N2 (n = 20) 8 (40.0) 12 (60.0) Tumor status T1/T2 (n = 47) 23 (48.9) 24 (51.1) 0.122 (d) T3/T4 (n = 13) 3 (23.1) 10 (76.9) Stage I/II (n = 37) 19 (51.4) 18 (48.6) 0.180 (d) III/IV (n = 23) 7 (30.4) 16 (69.6) KLK8-T4 Negative, Positive, Variable n (%) n (%) P Histotype ADC (b) (n = 33) 16 (48.5) 17 (51.5) 0.881 (c) SCC (n = 17) 7 (41.2) 10 (58.8) Other subtypes (n = 10) 5 (50.0) 5 (50.0) Tumor grade Poorly diff (n = 19) 7 (36.8) 12 (63.2) 0.417 (d) Diff (n = 39) 19 (48.7) 20 (51.3) Unknown (n = 2) Tumor size [less than or equal to] 3cm(n = 20) 9 (45.0) 11 (55.0) 1.000 (d) >3cm(n= 40) 19 (47.5) 21 (52.5) Nodal status N0 (n = 40) 19 (47.5) 21 (52.5) 1.000 (d) N1/N2 (n = 20) 9 (45.0) 11 (55.0) Tumor status T1/T2 (n = 47) 25 (53.2) 22 (46.8) 0.066 (d) T3/T4 (n = 13) 3 (23.1) 10 (76.9) Stage I/II (n = 37) 19 (51.4) 18 (48.6) 0.430 (d) III/IV (n = 23) 9 (39.1) 14 (60.9) (a) The cutoffs used were equal to the 38th, 43rd, and 47th percentiles for KLK8, KLK8-T3, and KLK8-T4 mRNAs, respectively. Statistically significant differences (P < 0.05) are in boldface. (b) ADC, adenocarcinoma; SCC, squamous cell carcinoma; diff, differentiated. (d) [chi square] test. (e) Fisher exact test. Table 3. Univariate and multivariate analysis of various prognostic factors in patients with NSCLC. (a) Cutoff Prognostic factor (percentile) Sex Age (>median vs <median) Histotype (ADC (b) vs SCC) Histotype (ADC vs other histotypes) Tumor size (>3cmvs [less than or equal to]3 cm) Differentiation (diff vs poorly diff) Stage (III/IV vs I/II) Smoking status mRNA production status (positive vs negative) KLK8 38 KLK8/KLK5 43 KLK8/KLK6 22 KLK8/KLK7 21 KLK8/KLK10 42 KLK8/KLK11 40 KLK8/KLK13 36 KLK8/KLK14 40 KLK8-T3 43 KLK8-T3/KLK5 46 KLK8-T3/KLK6 42 KLK8-T3/KLK7 38 KLK8-T3/KLK10 36 KLK8-T3/KLK11 40 KLK8-T3/KLK13 40 KLK8-T3/KLK14 53 KLK8-T4 47 KLK8-T4/KLK5 54 KLK8-T4/KLK6 43 KLK8-T4/KLK7 40 KLK8-T4/KLK10 42 KLK8-T4/KLK11 44 KLK8-T4/KLK13 50 KLK8-T4/KLK14 52 Univariate analysis Prognostic factor RR (95% CI) P Sex 0.53 (0.16-1.79) 0.309 Age (>median vs <median) 1.01 (0.97-1.05) 0.596 Histotype (ADC (b) vs SCC) 1.76 (0.74-4.19) 0.202 Histotype (ADC vs other histotypes) 1.62 (0.57-4.65) 0.367 Tumor size (>3cmvs [less than or equal to]3 cm) 1.08 (0.45-2.57) 0.867 Differentiation (diff vs poorly diff) 1.56 (0.71-3.42) 0.271 Stage (III/IV vs I/II) 4.14(1.84-9.33) 0.001 Smoking status 1.01 (0.99-1.02) 0.475 mRNA production status (positive vs negative) KLK8 2.56 (1.03-6.39) 0.043 KLK8/KLK5 2.22 (0.93-5.30) 0.073 KLK8/KLK6 0.79 (0.32-1.96) 0.606 KLK8/KLK7 2.99(0.89-10.02) 0.076 KLK8/KLK10 3.84 (1.50-9.81) 0.005 KLK8/KLK11 4.57 (1.68-12.46) 0.003 KLK8/KLK13 6.32 (1.87-21.31) 0.003 KLK8/KLK14 4.14(1.41-12.17) 0.010 KLK8-T3 2.64 (1.06-6.59) 0.037 KLK8-T3/KLK5 2.65 (1.11-6.32) 0.028 KLK8-T3/KLK6 2.53 (1.02-6.31) 0.046 KLK8-T3/KLK7 2.36 (0.95-5.88) 0.066 KLK8-T3/KLK10 3.58 (1.22-10.51) 0.020 KLK8-T3/KLK11 3.47 (1.29-9.34) 0.014 KLK8-T3/KLK13 4.25 (1.45-12.50) 0.009 KLK8-T3/KLK14 2.61 (1.12-6.10) 0.027 KLK8-T4 3.37 (1.35-8.41) 0.009 KLK8-T4/KLK5 3.06 (1.33-7.06) 0.009 KLK8-T4/KLK6 2.22 (0.93-5.30) 0.071 KLK8-T4/KLK7 2.74(1.10-6.85) 0.031 KLK8-T4/KLK10 4.97 (1.69-14.58) 0.004 KLK8-T4/KLK11 8.16(2.40-27.75) 0.001 KLK8-T4/KLK13 3.97 (1.56-10.09) 0.004 KLK8-T4/KLK14 3.94 (1.55-10.03) 0.004 Multivariate analysis Prognostic factor RR (95% CI) P Sex 0.41 (0.08-2.08) 0.282 Age (>median vs <median) 1.01 (0.97-1.06) 0.605 Histotype (ADC (b) vs SCC) 1.94 (0.64-5.87) 0.242 Histotype (ADC vs other histotypes) 2.13(0.51-8.91) 0.301 Tumor size (>3cmvs [less than or equal to]3 cm) 1.10 (0.40-3.02) 0.850 Differentiation (diff vs poorly diff) 1.80 (0.58-5.91) 0.307 Stage (III/IV vs I/II) 3.63 (1.53-8.60) 0.003 Smoking status 1.00 (0.97-1.02) 0.762 mRNA production status (positive vs negative) KLK8 1.45 (0.51-4.09) 0.486 KLK8/KLK5 1.67 (0.66-4.24) 0.283 KLK8/KLK6 0.45 (0.16-1.31) 0.143 KLK8/KLK7 1.94 (0.44-8.63) 0.386 KLK8/KLK10 4.20(1.38-12.83) 0.012 KLK8/KLK11 4.22 (1.32-13.52) 0.015 KLK8/KLK13 5.35 (1.24-23.02) 0.024 KLK8/KLK14 2.34 (0.67-8.10) 0.180 KLK8-T3 1.44 (0.51-4.05) 0.488 KLK8-T3/KLK5 1.53 (0.61-3.85) 0.370 KLK8-T3/KLK6 1.38 (0.49-3.89) 0.540 KLK8-T3/KLK7 1.25 (0.45-3.49) 0.672 KLK8-T3/KLK10 1.83 (0.53-6.39) 0.341 KLK8-T3/KLK11 2.63 (0.82-8.40) 0.103 KLK8-T3/KLK13 2.71 (0.73-10.15) 0.138 KLK8-T3/KLK14 1.74 (0.66-4.65) 0.264 KLK8-T4 3.90 (1.29-11.75) 0.016 KLK8-T4/KLK5 3.38(1.26-9.07) 0.015 KLK8-T4/KLK6 1.77 (0.70-4.46) 0.227 KLK8-T4/KLK7 5.51 (1.62-18.74) 0.006 KLK8-T4/KLK10 5.13 (1.45-18.09) 0.011 KLK8-T4/KLK11 8.37 (2.26-30.94) 0.001 KLK8-T4/KLK13 3.45 (1.17-10.15) 0.024 KLK8-T4/KLK14 3.33 (1.22-9.11) 0.019 (a) Statistically significant differences (P < 0.05) are highlighted in boldface. (b) ADC, adenocarcinoma; SCC, squamous cell carcinoma; diff, differentiated.
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|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Planque, Chris; Choi, Yun-Hee; Guyetant, Serge; Briollais, Nathalie Heuze-Vourch Laurent; Courty, Yv|
|Date:||Jun 1, 2010|
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