Liu et al. Journal of Translational Medicine 2013, 11:59 http://www.translational-medicine.com/content/11/1/59 RESEARCH Open Access Low expression of PTK6/Brk predicts poor prognosis in patients with laryngeal squamous cell carcinoma Xue-Kui Liu1,2†, Xin-Rui Zhang1,2†, Qian Zhong1,3, Man-Zhi Li1,3, Zhi-Min Liu1,2, Zhi-Rui Lin1,3,DiWu1,2 and Mu-Sheng Zeng1,3,4* Abstract Background: Protein tyrosine kinase 6 (PTK6), also known as breast tumor kinase (Brk), was a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. The deregulated expression of PTK6 was observed in various human cancers. However, little was known about PTK6 expression and its clinicopathological significance in human laryngeal squamous cell carcinoma (LSCC). Materials: PTK6 expression was evaluated in 7 pairs of surgically resectable laryngeal tissues by Western blotting and in 13 pairs of surgically resectable laryngeal tissues by reverse transcription-PCR (RT-PCR). Using immunohistochemistry, we performed a retrospective study of the PTK6 expression levels on 134 archival LSCC paraffin-embedded samples. Prognostic outcomes correlated with PTK6 were examined using Kaplan–Meier analysis and Cox proportional hazards model. Results: The PTK6 expression level was lower in LSCC tissues than in the adjacent noncancerous epithelial laryngeal tissues by Western blots and RT-PCR. By immunohistochemical analysis, we observed high expression of PTK6 in 25 of 76 (32.9%) adjacent noncancerous epithelial laryngeal tissues and in 39 of 134 (29.1%) of LSCC, respectively. Multivariate analysis demonstrated that pN status and the expression level of PTK6 (P < 0.05) were independent and significant prognostic factors. In the primary LSCC category, median DFS (disease free survival) of high, medium and low PTK6 expression patients were 88.5 months ,74.5 months and 49.0 months (log-rank test, P = 0.002); median OS (overall survival) of high, medium and low PTK6 expression patients were 88.5 months ,76.3 months and 65.7 months (log-rank test, P = 0.002). Reduced cytoplasmic PTK6 expression in LSCC was significantly associated with late pN status (P =0.005, r = 0.27), advanced pTNM stages (III and IV) (P =0.027, r = 0.147), and poor differentiated LSCC (P <0.0001, r = 0.486). In adjacent paracancerous laryngeal epithelial samples, median DFS of high, medium and low PTK6 expression patients were 92.6 months ,75.6 months and 48.5 months (log-rank test, P = 0.020); median OS of high, medium and low PTK6 expression patients were 92.9 months ,78.9 months and 74.6 months (log-rank test, P = 0.042). Conclusion: The present findings indicated that cytoplasmic PTK6 expression is a potential prognostic factor for survival in LSCC patients. High expression of PTK6 was associated with favorable OS and DFS in LSCC patients. Keywords: PTK6/Brk, Laryngeal squamous cell carcinoma, Prognosis * Correspondence: [email protected] †Equal contributors 1State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Centre, Guangzhou, Guangdong, China 3Department of Experimental Research, Sun Yat-Sen University Cancer Centre, Guangzhou, Guangdong, China Full list of author information is available at the end of the article © 2013 liu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Liu et al. Journal of Translational Medicine 2013, 11:59 Page 2 of 9 http://www.translational-medicine.com/content/11/1/59 Background (MRI), bilateral cervical and supraclavicular ultrasonog- The cytoplasmic non-receptor tyrosine kinase PTK6 raphy, and abdominal ultrasonography. The medical re- (BRK, breast tumour kinase) was originally cloned from a cords of these patients were reviewed to assess the human metastatic breast tumour [1]. The 53 kDa PTK6 patients’ characteristics, including age, sex, primary site, protein was a cytoplasmic tyrosine kinase, and structurally clinical stage, date of disease progression, and final sta- resembled tyrosine kinases of the Src family [2]. Src family tus on the last follow-up examination. The informed tyrosine kinases play important roles in epithelial tumor consent was obtained from each patient prior to surgery development. Therefore, it was proposed that PTK6 may and the study was approved from the Institute Research play a role in epithelial tumorigenesis [3,4]. Ethics Committee. The overexpression of tyrosine kinases (including EphA1, PTK6/BRK, and Ron) were reported in head and Western blot neck cancers which included pharyngeal, hypopharyngeal, Western blotting analysis was carried out with the pro- tonsilar, supraglottic and some oral cancers [5]. However, teins collected from the adjacent normal epithelium tis- PTK6 expression in tumor tissues was complex. For ex- sues and the cancer tissues, and total proteins were ample, PTK6 was localized in the nucleus and cytoplasm extracted with 1 X sodium dodecyl sulfate (SDS) sample of normal oral epithelium, and in perinuclear regions of buffer (2% SDS, 62.5 mmol/L Tris–HCl (pH 6.8), 5% 2- poorly differentiated oral squamous carcinomas [3]. PTK6 mercaptoethanol, and 10% glycerol) . The concentration expression was low or undetectable in normal ovary [2] or of the protein was measured by the BCA protein assay normal breast epithelium [3,4,6], but it was found in hu- kit (PIERCE, Rockford, IL, USA). A total of 20 μg pro- man ovarian tumor cells [2] and in greater than 60% of tein was electrophoretically separated in 12% SDS poly- breast tumors and breast cancer derived cell lines [7], indi- acrylamide gels and transferred onto polyvinylidene cating that overexpression of PTK6 may be related to car- difluoride membranes (Amersham Pharmacia Biotech, cinogenesis. Aubele et al. [6] reported that PTK6 protein Piscataway, NJ). Then incubate the primary polyclonal expression had prognostic value in a small set of 105 antibody against PTK6 (dilution, 1:1000; Abgent INC, breast carcinomas. However, the expression of PTK6 and USA),which was mixed with PTK6 Antibody (N-term) its clinical significance were not clearly documented in Blocking Peptide (0.25 ug/ml ), in 5% of the skimmed LSCC. In the current study, we aimed to investigate PTK6 milk solution overnight at 4°C, and anti-rabbit (1:3000; expression and analyze its association with clinicopatho- Santa Cruz Biotechnology, Santa Cruz, CA) secondary logical factors to understand its potential role in LSCC. antibody was were used to detect PTK6 protein. Anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Material and method (1:4000, Santa Cruz, CA, USA) antibody and anti-mouse Tissue samples and patients (1:4000; Santa Cruz Biotechnology, Santa Cruz, CA) sec- Fresh tumor tissue samples with paired non-cancerous ondary antibody were used to confirm equal loading. normal mucosa (with more than a 5-mm distance from The protein signals were detected by the enhanced the primary tumor’s edge) of 13 LSCC patients were chemiluminescence (ECL) detection system (Amersham obtained at the time of operation from the Sun Yat-sen Biosciences Europe, Freiburg, Germany) according to University Cancer Center (SYSUCC). All the 13 patients the manufacturer's protocols. were histologically confirmed as LSCC by biopsy pre- operation and all these patients underwent total laryn- Real time reverse transcription-polymerase chain reaction gectomy in the primary site. Before making extraction, (RT-PCR) analysis we did frozen section and made sure that all the speci- Total RNAs from fresh tissues were purified from tissues mens were done on at least 70%-80% tumor cells under using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) the microscope. A total of 134 patients who were previ- according to the manufacturer’s instructions, and 2 μg ously untreated and histologically confirmed LSCC, in RNA of each sample was reverse transcribed using resectable stages (T1~4aN0-3 M0 ) (Union for Inter- SuperScript RT kit (Invitrogen Life Technologies, Carls- national Cancer Control ,UICC 2002) were eligible be- bad, CA, USA). Full-length open reading frame of PTK6 tween January 2003 and December 2005 at SYSUCC in was amplified by PCR from cDNA samples of normal la- our study. Patients who had previous malignant disease, ryngeal epithelium tissues and laryngeal carcinoma tis- a second primary tumor, positive margin, or died of sues. Real-time PCR was carried out using a CFX96 postoperative complications were excluded. Pretreat- Real-Time System (BIO-RAD) (PREMIER Biosoft ment evaluations included a complete history, physical International, Palo Alto CA, USA). Sequences of the examination, performance status, serum chemistry pro- primers were as follows: PTK6, 5’-TACTTTGGGGA file, complete blood cell count, chest radiography, com- GGT CTTCGAG-3’(sense), 5’-TGCCGCAGCTTCTTC puted tomography (CT) or magnetic resonance imaging ATG-3’(antisense);GAPDH, 5'-GACTCATGACCACAG Liu et al. Journal of Translational Medicine 2013, 11:59 Page 3 of 9 http://www.translational-medicine.com/content/11/1/59 TCCATGC-3' (sense), 5'-AGAGGCAGGGATGATGTT LSCC patients were divided into three groups (1–2, low CTG-3' (antisense). We used the SYBR Green kit (Invitrogen expression; 3–5, medium expression and 6–7, high Life Technologies, Carlsbad, CA, USA) to execute the PTK6 expressing). Each section was evaluated by two in- amplification of the cDNA. The RT-PCR cycling pa- dependent pathologists without knowledge of the clinical rameters were performed as follows: denaturation at features of the cases. 95°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds. The expression Statistical analysis data were normalized to the geometric mean of house- The paired T-test was used to analyze the significance of keeping gene GAPDH to control the difference in ex- PTK6 protein levels and mRNA levels in the paired sam- pression levels and analyzed using the 2-Delta Delta C ples.