Prognostic significance of phosphorylated RON in esophageal squamous cell carcinoma

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1 DOI /s ORIGINAL PAPER Prognostic significance of phosphorylated RON in esophageal squamous cell carcinoma Marco K. C. Hui Kenneth K. Y. Lai Kwok Wah Chan John M. Luk Nikki P. Lee Yvonne Chung Leo C. Cheung Gopesh Srivastava Sai Wah Tsao Johnny C. Tang Simon Law Received: 7 October 2011 / Accepted: 2 November 2011 Ó Springer Science+Business Media, LLC 2011 Abstract Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer. RON is a transmembrane receptor overexpressed in various cancers; however, the clinical significance of its phosphorylated form () is not fully deciphered. This report is the first to investigate the expression and clinical significance of in human ESCC. Quantitative polymerase chain reaction revealed an up-regulation of RON mrna in 70% (7/10) of ESCC tissues when compared to the adjacent Marco K. C. Hui and Kenneth K. Y. Lai contributed equally to this work. Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. M. K. C. Hui K. K. Y. Lai N. P. Lee Y. Chung L. C. Cheung S. Law (&) Department of Surgery, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong slaw@hkucc.hku.hk K. W. Chan G. Srivastava Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong J. M. Luk Department of Pharmacology and Surgery, National University Health System, Singapore, Singapore S. W. Tsao Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong J. C. Tang Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Pokfulam, Hong Kong nontumor tissues. An overexpression of protein was found in most of the ESCC cell lines studied (4/5) when compared to two non-neoplastic esophageal epithelial cells using immunoblot. In 64 ESCC tissues, was localized at the cell membrane, cytoplasm and nucleus in 15 (23.4%), 63 (98.4%) and 61 (95.3%) cases using immunohistochemistry. Patients having high expression of cytoplasmic significantly associated with shorter median survival when compared to those with low expression (25.41 months vs months), suggesting cytoplasmic as a potential marker for poor prognosis in ESCC patients. Keywords Esophageal squamous cell carcinoma Phosphorylated RON Overexpression Poor prognostic marker Median survival after surgery Introduction Esophageal cancer is the eighth most prevalent cancer worldwide and ranks sixth as the most common cause of cancer-related deaths. Among all histological subtypes, esophageal squamous cell carcinoma (ESCC) is the predominant type in Asia. Despite various therapeutic options such as surgery, chemotherapy and radiotherapy, the prognosis of ESCC is relatively poor with the overall 5-year survival rate at 10 40% [1, 2]. Recepteur d origine nantais (RON) is a 185 kda heterodimeric transmembrane glycoprotein composed of a 35 kda extracellular a-chain and a 150 kda transmembrane b-chain [3]. Upon ligand binding, RON is activated via autophosphorylation within its kinase catalytic domain, resulting in an array of effects, including increase in cellular motility, invasiveness and apoptosis, which are

2 important features of tumor metastasis [4 6]. RON expression is often altered in human cancers like those happened in colon, bladder and pancreas [5, 7 12]. Most of these studies focus on studying the expression and involvement of RON and its splice variant, but not the phosphorylated RON (), in tumorigenesis. To address this, we investigated the expression of in ESCC and examined its prognostic significance. Materials and methods Human esophageal cell lines Five human ESCC cell lines (HKESC-1, HKESC-2, HKESC-3, HKESC- 4 and SLMT-1) and two human nonneoplastic esophageal cell lines (NE-1 and NE-3) were used as described, for which these cell lines were established by our team earlier [13 18]. Both NE-1 and NE-3 cells were cultured in Keratinocyte-SFM medium (Invitrogen, Carlsbad, CA) supplemented with bovine pituitary extract and human recombinant EGF. HKESC-1 and HKESC-4 were maintained in Minimum Essential Medium (MEM) medium (Invitrogen) with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen) and 1% penicillin and streptomycin (P/S) (Invitrogen). HKESC-2, HKESC-3 and SLMT-1 cells were cultured in MEM medium with 20% FBS and 1% P/S. Patient specimens We have recruited 64 ESCC patients who had undergone esophagectomy, without neoadjuvant chemotherapy nor radiotherapy, between 1997 and 2005 at Queen Mary Hospital, Hong Kong. Tumor and nontumor tissues near the proximal resection margins were obtained and stored at -80 C for subsequent analyses; 5 lm of paraffin embedded sections were prepared and stained with hematoxylin and eosin for light microscopy, which were then classified into different grades based on the criteria of the World Health Organization. Cancers were staged according to the TNM classification. GG-3 0 and reverse: 5 0 -GCG ATG CTG TTG AAC ACT-3 0 ), following the protocol as described [19, 20]. Quantification of the gene expression was performed in ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA). Immunoblot Immunoblotting was performed as described [21, 22]. Rabbit polyclonal antibody (1:1,500) (Santa Cruz Biotechnology, Santa Cruz, CA) was used as the primary antibody. b-actin antibody (1:5,000) (Sigma-Aldrich, St Louis, MO) was used as an internal control. Goat antirabbit (1:7,000) (Zymed, Carlsbad, CA) or goat anti-mouse (1:20,000) (Zymed) antibodies conjugated with horseradish peroxidase (HRP) was used for detecting or b-actin, respectively. Protein expressions were visualized using ECL Plus Western blotting reagent pack (GE Healthcare Biosciences, Piscataway, NJ) followed by radiography. Immunohistochemistry Immunohistochemistry was performed as described [23, 24]. In brief, 10% normal goat serum (in TBS) (Dako, Glostrup, Denmark) was used to block the nonspecific (a) 25 RON expression level (b) NE NE HKESC-1 HKESC HKESC-3 HKESC Patients SLMT Reverse transcription (RT) and quantitative polymerase chain reaction (qpcr) β-actin 185 kda 150 kda 42 kda TRIzol reagent (Invitrogen) was used for RNA extraction in ESCC specimens, according to manufacturer s instructions. RT was started with 500 ng RNA using SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen), following manufacturer s protocol; 3 6 lg cdna was subjected for qpcr using Platinum Quantitative PCR SuperMix-UDG w/rox (Invitrogen) and RON-specific primers (forward: 5 0 -AAG GAC CTC ATC AGC TTT Fig. 1 High level of RON mrna in ESCC tissues and protein in ESCC cells. a qpcr showed high level of RON mrna in ESCC tissues when compared to adjacent nontumor tissues of the same patient in 7 out of 10 cases. The fold ratio of RON mrna in nontumor tissue was arbitrarily set to 1 in all cases. b Immunoblotting data showed high protein level of in most ESCC cell lines (HKESC-1, HKESC-2, HKESC-4 and SLMT-1) when compared to non-neoplastic esophageal cell lines (NE-1 and NE-3). b-actin was used as a loading control. Each sample was repeated in at least duplicates

3 binding sites following by incubating antibody (1:600). Anti-rabbit labeled polymer-hrp provided in EnVision? System-HRP (Dako) was then used. Signals were visualized by incubating with liquid DAB? (diaminobenzidine) (Dako) before counterstaining with hematoxylin. Negative controls were performed using normal goat serum instead of antibody. staining was evaluated by Dr. KW Chan, a qualified pathologist, under light microscope. According to Wang et al. [25] with modifications, a weighted index score (0 9) Fig. 2 High specificity of antibody. antibody yielded positive signals when used for immunoblot (a) and immunohistochemistry (b). However, no signals were observed when the antibody was first pre-incubated with an excess amount of blocking peptide (a, c). Original magnification, 9100 (b) (a) Corresponding blocking peptide - + (c) 185 kda 150 kda Fig. 3 in ESCC tissues. antibody was used to detect in ESCC tissues using immunohistochemistry, and positive signal was observed (a). No signal was observed when normal goat serum was used to replace the antibody in parallel experiment (b). Original magnification, ESCC tissues exhibit varied expression of, such that low (c) or high (d) expression of was detected in different specimens. Various localizations of were observed in different ESCC specimens, such that was found associating either with membrane (indicated by an arrow) (e) or nucleus (indicated by an arrow) (f). Original magnification, 9100

4 was calculated for each tumor by multiplying the values of these two categories, that is, low expression group with weighted index score 0 4 and high expression group with weighted index score 5 9. The cellular expression of at the cytoplasm and nucleus was also determined. Prediction of nuclear localization signal (NLS) PSORT II software [26] ( was utilized to predict any putative NLSs in human RON peptide sequence (NCBI accession number: NP_002438). Statistical analyses Data were expressed as mean ± SEM. Pearson s Chi-square test was used to assess the correlations between expression and various clinicopathological parameters. Kaplan Meier method was employed for analyzing survival, and the differences in survival were estimated using log-rank test. Any parameter with a P value\0.05 in the univariate analysis was subjected for multivariate analysis. A P value \0.05 was considered statistically significant. Statistical analyses were performed using SPSS 12.0 for Windows (Chicago, IL). Results High expression of RON in ESCC tissues qpcr results showed an up-regulation of RON mrna in 70% (7 of 10) ESCC tissues when compared to the corresponding nontumor tissues (Fig. 1a). The fold change of RON expression in ESCC tissues versus nontumor tissues ranged from *3-fold (Patient 10) to *23-fold (Patient 6). ESCC tissues from Patient 4 and 5 showed down-regulation of RON mrna. ESCC tissue from Patient 1 had no apparent difference in the expression of RON. High expression of in ESCC cell lines Immunoblot was used to determine the protein level of in esophageal cell lines. The antibody used was able to detect intact RON of 185 kda (with both a and b chains) and cleaved RON of 150 kda (with b chain only). To ensure the specificity of the staining, preincubation of the antibody with its blocking peptide was performed and no detectable signal was observed (Fig. 2a). Similar observation was noticed when this antibody was pre-incubated with blocking peptide before immunohistochemistry (Fig. 2b, c). Using this antibody for immunoblot, most ESCC cells (HKESC-1, HKESC-2, Fig. 4 in non-neoplastic esophageal squamous tissue. was found densely in the proliferating layer of the esophageal epithelium using immunohistochemistry. Original magnification, 9200 HKESC-4 and SLMT-1) except HKESC-3 showed an up-regulation of level, while the two non-neoplastic cell lines NE-1 and NE-3 expressed minimal level of this protein (Fig. 1b). Low expression of cytoplasmic associates with better survival in ESCC patients Immunohistochemistry was used to examine the cellular expression of in 64 ESCC tissues and 10 randomly selected nontumor tissues. Negative control utilizing normal goat serum instead of the antibody yielded no immunoreactivity (Fig. 3b). Expression of was detected at cell membrane, cytoplasm and nucleus in ESCC tissues (Fig. 3). Membranous, cytoplasmic and nuclear were found in 23.4% (15/64), 98.4% (63/64) and 95.3% (61/64) of ESCC tissues, respectively. was associated preferentially in the proliferating layer of the non-neoplastic epithelium and not found in the upper and more differentiated layer in the nontumor tissues (Fig. 4). No correlation between cytoplasmic (Table 1) or nuclear (Table 2) with any clinicopathological parameters was identified. The statistical analysis of membranous with clinicopathological parameters was not attempted due to the small number of samples with membranous. Cytoplasmic expression was associated with patients survival in univariate analysis (log-rank test: P = 0.034, Fig. 5a), while nuclear was not (logrank test: P = 0.164, Fig. 5b). Patients with low expression of cytoplasmic had a longer median survival (25.41 months, 95% CI: months) when

5 Table 1 Correlations between cytoplasmic expression and various clinicopathological parameters in ESCC patients Subgroups Clinicopathological parameters P value Table 2 Correlations between nuclear expression and various clinicopathological parameters in ESCC patients Subgroups Clinicopathological parameters P value Low expression of cytoplasmic High expression of cytoplasmic Low expression of nuclear High expression of nuclear Age (grouped) Below or equal Above Gender Female Male Smoking Nonsmoker Smoker Level of tumor Upper Middle Lower 9 5 Double 2 1 Tumor differentiation Poor Moderate Well 8 4 R category R R1/R T-stage Early (T1/T2) Advanced (T3/T4) N-stage N N M-stage M M1a/M1b 4 2 Overall pathological stage Early (stage I/II) Advanced (stage III/IV) Age (grouped) Below or equal Above Gender Female Male 40 7 Smoking Nonsmoker Smoker 28 5 Level of tumor Upper Middle 31 6 Lower 13 1 Double 3 0 Tumor differentiation Poor Moderate 32 6 Well 10 2 R category R R1/R T-stage Early (T1/T2) Advanced (T3/T4) 45 6 N-stage N N M-stage M M1a/M1b 5 1 Overall pathological stage Early (stage I/II) Advanced (stage III/IV) 34 5 compared to those patients with high expression (14.43 months, 95% CI: months). In multivariate analysis (Table 3), cytoplasmic significantly correlated with patients survival (cox regression: P = 0.004, RR: and 95% CI: ). R category was also significant (P = 0.009, RR: and 95% CI: ), and N-stage was marginally significant (P = 0.051, RR: and 95% CI: ) in correlating with patients survival. Presence of NLSs in human RON Due to the presence of nuclear in ESCC tissues, we employed PSORT II to identify any putative NLSs present in human RON that enable the nuclear translocation of. Five putative NLSs were found in RON peptide sequence. Four of them were located at the amino-terminus: (1) PKRR, amino acid ; (2) KRRR, amino acid ; (3) RRRR, amino acid ; and (4) PKRRRRG, amino

6 Fig. 5 Kaplan Meier survival curves for cytoplasmic and nuclear. Survival analysis was performed for cytoplasmic in a cohort of 25 high expression patients and 39 low expression patients. Log-rank test: P = (a). Survival analysis was performed for nuclear in a cohort of 9 high expression patients and 55 low expression patients. Log-rank test: P = (b) acid One NLS was found at the carboxyl-terminus: RRPR, amino acid 1,388 1,391 (Supp. fig. 1). Discussion This is the first study to report the prognostic significance of cytoplasmic in ESCC by stating patients with low cytoplasmic expression associate with longer median survival after surgery when compared to those with high expression. To our knowledge, no immunohistochemical analysis using -specific antibody was performed to investigate its prognostic importance in any types of cancer. Instead, several studies demonstrated the prognostic significance of total RON expression (i.e., the nonphosphorylated RON and ) in bladder carcinoma [8, 11], breast cancer [27], pancreatic cancer [7, 12] and renal cancer [28] using antibodies capable of detecting both forms. Here, we used -specific antibody to provide the first insight on the clinical correlation of in ESCC. RON is a transmembrane receptor, thus it is expected for its localization at the cell membrane. However, cytoplasmic expression of RON was reported in several cancers like pancreatic cancer using immunohistochemistry, and no reason has been given to explain for this observation [7, 12]. In addition, we have demonstrated here for the presence of in both cytoplasm and nucleus of ESCC tumors. Therefore, it is speculated for RON to behave like other membrane receptors, such as androgen receptor [29], ErbB4 [30] and epidermal growth factor receptor (EGFR) [31], in relocating to other cellular compartments. Indeed, c-met receptor, which also belongs to the same family of RON, is also capable of translocating to the cell nucleus [32]. It is proposed that nuclear expression of these membrane receptors could activate gene transcription [33, 34],

7 Table 3 Correlations between ESCC patients survival after surgery and various clinicopathological parameters Subgroups Clinicopathological parameters Univariate analysis Multivariate analysis RR (95% CI) P value RR (95% CI) P value Age (n = 64) Below or equal Above ( ) Gender Female Male ( ) Smoking Nonsmoker Smoker ( ) Level of tumor Upper 1 Middle ( ) Lower 0.61 ( ) Double ( ) Tumor differentiation Poor 1 Moderate ( ) Well ( ) R category R R1/R ( ) ( ) T-stage Early (T1/T2) Advanced (T3/T4) ( ) N-stage N N ( ) ( ) M-stage M M1a/M1b ( ) Overall pathological stage Early (stage I/II) Advanced (stage III/IV) ( ) ( ) Cytoplasmic Low High ( ) ( ) which, therefore, indicates for the roles of these receptors in enhancing signaling pathway at the transcriptional level [31]. NLS is a stretch of basic amino acids within a peptide sequence, which directs host proteins to the cell nucleus via interacting with the nuclear transport receptor importin [35]. In this study, we have used bioinformatics to show the presence of five putative NLSs in human RON peptide sequence. The presence of these NLSs implicates for the potential of in translocating into cell nucleus as observed in this study. This also helps to explain for the presence of cytoplasmic. However, further experiments are required to validate this notion. Although an overexpression of RON associates with increase in cell motility and protects cells against apoptosis [36], the functions of nonmembranous RON remain to be identified.

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