J Mol Hist (2012) 43:715–721 DOI 10.1007/s10735-012-9438-7

ORIGINAL PAPER

Down-regulation of tumor suppressor in lung cancer 1 (TSLC1) expression correlates with poor prognosis in patients with colon cancer Jinfeng Zhang • Jinfeng Ning • Jingshu Geng Binbin Cui • Xinshu Dong

•

Received: 7 June 2012 / Accepted: 22 July 2012 / Published online: 1 August 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Tumor suppressor in lung cancer 1 (TSLC1), a novel tumor suppressor gene, has been reported to be frequently inactivated in a variety of human malignant tumors. The aim of this study was to detect TSLC1 expression in human colon cancer and to analyze its association with prognosis of patients with colon cancer. Using quantitative real-time PCR and Western blot analysis, we found significantly decreased expression of TSLC1 in primary colon tumor tissues (n = 30) compared with adjacent normal tissues. Immunohistochemistry analysis also found decreased TSLC1 expression in 41.3 % (33/80) colon tumor tissues. In clinicopathological analysis, loss of TSLC1 expression significantly correlated with female gender and lymph node metastasis of colon cancer patients (P \ 0.05). In addition, decreased expression of TSLC1 in tumors was found to be closely associated with a poor prognosis (P = 0.037, log-rank test), and multivariate

J. Zhang  J. Ning Department of Thoracic Surgery, The Third Affiliated Hospital of Harbin Medical University, Harbin 150081, People’s Republic of China J. Zhang  X. Dong (&) Department of Oncology, The Fourth Affiliated Hospital of Harbin Medical University, 37 Yiyuan Street, Harbin 150001, People’s Republic of China e-mail: [email protected] J. Geng Department of Pathology, The Third Affiliated Hospital of Harbin Medical University, Harbin 150081, People’s Republic of China B. Cui Department of Gastrointestinal Surgery, The Third Affiliated Hospital of Harbin Medical University, Harbin 150081 People’s Republic of China

analysis showed that lower TSLC1 protein expression was an independent prognostic factor for colon cancer patients. Our study suggests that down-regulated expression of TSLC1 may play an important role in the progression of colon cancer and TSLC1 expression may serve as a useful marker for the prognostic evaluation of patients with colon cancer. Keywords Tumor suppressor in lung cancer 1  Colon cancer  Prognosis  Human

Introduction Colorectal cancer (CRC) is the second most common human malignant disease and the fourth most frequent cause of cancer-related mortality, with an estimated of 1.2 million new cases and 0.6 million deaths worldwide in 2008 (Jemal et al. 2011). Despite curative surgical removal of the primary tumor, a significant portion of patients with CRC suffer recurrence and die within 5 years (Punt and Tol 2009; Hong et al. 2010). Unfortunately, there are no available molecular markers that provide reliable prognostic information for patients following surgical resection of CRC. Thus, there is an acute need for novel molecular biomarkers capable of serving as prognostic markers for clinical CRC. Tumor suppressor in lung cancer 1 (TSLC1), a novel tumor-suppressor gene candidate, was initially identified by Murakami et al. using the combinatorial analyses of yeast artificial chromosome in human non-small cell lung cancer (NSCLC) and tumorigenicity assay in nude mice (Kuramochi et al. 2001). TSLC1 encodes a single-pass transmembrane glycoprotein belonging to the immunoglobulin superfamily and participates in calcium-independent

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Table 1 Association of TSLC1 mRNA with protein in colon cancer tissues TSLC1 mRNA

TSLC1 protein

High or unchanged

Low

High or unchanged

11

1

Low

2

16

P value

j

0.000

0.795

cell–cell adhesion and cell signal transduction (Masuda et al. 2002). Previous studies have shown that TSLC1 is ubiquitously expressed in a wide range of human tissues, such as lung, brain, testes as well as most epithelial and neuronal tissues (Liang et al. 2011). Recently, TSLC1 protein expression is found to be lost or down-regulated in a variety of human cancers, including lung (Goto et al. 2005), pancreatic (Jansen et al. 2002), breast (Heller et al. 2007; Takahashi et al. 2012), nasopharyngeal (Lung et al. 2006), cervical (Yang et al. 2006), and ovarian cancer (Yang et al. 2011). In addition, Chen and colleagues have reported that epigenetic inactivation of TSLC1 by promoter hypermethylation is frequently observed in CRC and correlates with the advanced stage of the disease (Chen et al. 2011). However, the prognostic value of TSLC1 expression in colon cancer has not yet been investigated. In the present study, we evaluated TSLC1 expression in colon cancer tissues and analyzed its correlation with clinicopathological parameters of colon cancer patients. In addition, we also investigated whether TSLC1 expression could be a molecular marker for predicting the outcome of colon cancer patients.

Materials and methods Patients and tissue samples One hundred and ten tissue samples including 80 primary tumor lesions and 30 corresponding adjacent normal colon tissues were consecutively collected from patients with colon cancer who underwent curative surgical resection at the Department of Gastrointestinal Surgery, The Third Affiliated Hospital of Harbin Medical University between December 2004 and December 2005. The patients consisted of 45 men and 35 women with a mean age of 65 years and a range of 41–93 years. None of the patients received radiotherapy, chemotherapy, or other medical interventions prior to surgical operation. The clinicopathological findings were determined according to the classification of malignant tumors by the World Health Organization and International Union against Cancer

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Tumor-Node-Metastasis (TNM) staging system (Sobin and Fleming 1997). The clinicopathological variables were obtained by a medical history review and are summarized in Table 1. All patients were followed from the excision of the primary tumor until death, or the closing date of this study (December 17, 2010). The average follow-up for all patients was 54 months (range, 2–68 months). Sixty fresh specimens including 30 paired tumor tissues and corresponding normal tissues were frozen in liquid nitrogen immediately after surgical resection for extraction of RNA and protein. Each patient signed a written informed consent form before surgery, and the study protocol was reviewed and approved by the Institutional Review Board for the Use of Human Subjects at Harbin Medical University. Immunohistochemistry Immunohistochemistry was performed according to the standard streptavidin peroxidase complex technique. Briefly, colon cancer tissues were fixed in 10 % neutral buffered formalin overnight, embedded in paraffin, and sectioned with a thickness of 5 lm. The sections were given thermal pre-treatment at 60 °C for 2 h, dewaxed in xylene, and re-hydrated in an ethanol gradient. After antigen retrieval with autoclave treatment in 10 mmol/L citrate buffer (pH 6.0), endogenous peroxidase activity was quenched by incubating the sections in 0.3 % H2O2 for 15 min at room temperature. Subsequently, the sections were treated with 10 % normal goat serum to eliminate non-specific protein binding, and then incubated at 4 °C overnight with a rabbit polyclonal antibody against human TSLC1 (1:100 diluted; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After rinsing in phosphate-buffered saline (PBS), the sections were sequentially incubated with streptavidin-biotinylated anti-rabbit immunoglobulin G (IgG) as the secondary antibody, followed by incubation for 30 min with streptavidin horseradish peroxidase conjugate. Finally, reaction products were visualized by using 3, 30 -diaminobenzidine (DAB), and the sections were then counterstained with hematoxylin. Negative controls were achieved by an isotype matched IgG in each of the immunostaining. Evaluation of immunohistochemical staining Immunostaining results were determined separately by three investigators who were unaware of the patients’ identity or clinical status. In discrepant cases, a consensus was obtained via their simultaneous examination. TSLC1 expression was evaluated using a semi-quantitative scoring system where both intensity and percentage of positive tumor cells were analyzed (Yang et al. 2012; Chen et al. 2012). The intensity of staining was scored as 0 for

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negative, 1 for weak, 2 for moderate, and 3 for strong positive. The percentage of positive tumor cells was scored as 0 for 0 %, 1 for 1–10 %, 2 for 11–50 %, and 3 for 51–100 %. The sum of both scores was used to identify two categories of expression: low expression (with a total score B2) and high expression (with a total score C3). Quantitative real-time PCR Total RNA was isolated from the fresh tumor and non-tumor tissues using the RNA simple total-RNA kit (Tiangen, Beijing, China), and the purity of RNA was determined by measuring the absorbance at 260 and 280 nm with a spectrophotometer. One microgram of total RNA from each sample was reverse-transcribed to single-strand cDNA with an oligo dT primer. Quantitative real-time PCR reactions were performed in triplicate on an ExicyclerTM 96 thermal block (Bioneer, Daejeon, Korea) using the Real Master Mix SYBR green kit (Tiangen, Beijing, China). The thermal cycling program was set as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Post-amplification melting curves were generated to assure product specificity. The forward and reverse primers were 50 -ATGATGAAATGCCTCAACACGC-30 and 50 - TCCGA GTGAGCTTTCCCCACTA -30 for the target gene TSLC1 and were 50 - CTTAGTTGCGTTACACCCTTTCTTG-30 and 50 -CTGTCACCTTCACCGTTCCAGTTT-30 for the reference gene b-actin. Data were analyzed through the comparative threshold cycle (CT) method (Livak and Schmittgen 2001). Western blot analysis Total proteins were extracted from frozen tissues using ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of proteins were separated on 10 % sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), followed by a transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) at 70 V for 1.5 h. After blocking with 5 % non-fat dry milk, the membranes were probed at 4 °C overnight with rabbit anti-human polyclonal TSLC1 antibody (1:1000 diluted; Santa Cruz Biotechnology) or mouse antihuman monoclonal b-actin antibody (1:5000 diluted; Sigma, St. Louis, MO, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were visualized with enhanced chemiluminescence (ECL; Millipore) and exposed on X-ray films.

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Statistical analysis All statistical analyses were conducted using the SPSS 13.0 software package (SPSS Inc., Chicago, IL, USA). The Chi-square (v2) test was used for comparison between categorical variables and Student’s t test for parametric continuous variables. The Kappa test was used to assess the coincidence of TSLC1 mRNA and protein expression in colon cancer tissues. Survival curves were plotted by the Kaplan–Meier method and compared using the logrank test. Cox proportional hazards models were utilized for univariate and multivariate analyses. The hazard ratio (HR) with 95 % confidence interval (CI) and P values were reported. All tests were two-sided. The values of P less than 0.05 were considered statistically significant.

Results Expression of TSLC1 mRNA and protein in colon cancer specimens Firstly, we detected the expression of TSLC1 mRNA in 30 paired colon cancer tissues and their corresponding nontumor tissues by quantitative real-time PCR. We found that TSLC1 mRNA expression was detectable in all the colon cancer and corresponding non-tumor tissues, whereas the levels of TSLC1 expression in colon cancer tissues were much lower than those in corresponding non-tumor tissues (P \ 0.01, Fig. 1a). Moreover, 17 of 30 (56.7 %) analyzed samples exhibited a lower level of TSLC1 mRNA in colon cancer tissues than in their corresponding non-tumor tissues. To investigate whether TSLC1 down-regulation was also apparent at the protein level, we performed Western blot analysis of TSLC1 protein in these 30 paired samples. As expected, changes in 27 paired tissues (27/30, 90.0 %) observed by Western blot analysis were in agreement with the observations in the quantitative realtime PCR analysis, including low-level in 16 tumors and high-level or unchanged in 11 tumors. Representative blots are shown in Fig. 1b. Statistical analysis also indicated that the protein expression levels of TSLC1 were significantly down-regulated in tumor tissues compared with their normal counterparts (Fig. 1c, P \ 0.05). The Kappa test revealed a significant correlation between the expression of TSLC1 mRNA and protein (Table 1, P = 0.000, j = 0.795). Collectively, our data suggest that reduced expression of TSLC1 plays important roles in the development and progression of colon cancer.

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58.7 % (47/80) of primary colon cancer tissues were classified as high-level TSLC1 expression and 41.3 % (33/ 80) of the samples were classified as low-level TSLC1 expression. Table 2 summarizes the relationship between TSLC1 expression and clinicopathological parameters of colon cancer patients. No significant associations were found between TSLC1 expression and age, tumor differentiation, tumor size, and TNM stage (P [ 0.05). Interestingly, statistical analysis revealed that low-level TSLC1 expression was strongly associated with female gender (P = 0.042) and lymph node metastasis (P = 0.016). Relationship between TSLC1 down-regulation and prognosis of patients with colon cancer

Fig. 1 TSLC1 mRNA and protein expression primary colon cancer and corresponding normal tissues detected by quantitative real-time PCR (a) and Western blot analysis (b), respectively. a Data were normalized to expression of b-actin. b Representative blots of three independent experiments are shown, and the protein size is expressed in kDa. c Densitometric values were normalized by b-actin levels

Correlations of TSLC1 protein expression with clinicopathological characteristics of colon cancer patients To further evaluate the clinical relevance of TSLC1 expression in colon cancer, we examined the expression of TSLC1 protein in 80 colon cancer tissue samples by using immunohistochemistry. As illustrated in Fig. 2, immunoreactivity of TSLC1 protein was observed primarily in the cytoplasm of tumor cells in colon cancer tissues. Overall,

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All patients were follow-up for up to 68 months, with a mean survival time of 54 months. Seventeen patients died of recurrence during the postoperative follow-up period. In order to investigate the prognostic value of TSLC1, we analyzed the relationship between TSLC1 protein expression and patient survival. Kaplan–Meier analysis showed that patients with high TSLC1 expression had longer survival times, whereas those with low TSLC1 expression had shorter survival times (Fig. 3, log-rank, P = 0.037). The cumulative 5-year survival rate was 62.9 % (95 % CI, 0.590–0.669) in the high TSLC1 group, compared to only 56.1 % (95 % CI, 0.497–0.626) in the low TSLC1 group. To further identify the variables of potential prognostic significance in the patients with colon cancer, univariate analysis of each variable was performed in relation to the survival time. The difference in predicting the prognosis was assessed by examining the HR and P-value for each variable. By univariate analysis, factors including the TNM stage and TSLC1 protein expression showed a significantly higher HR for a poor prognosis. Additionally, multivariate analysis revealed that TSLC1 expression was recognized as an independent prognostic factor of patient outcome (Table 3). Taken together, our results indicate that TSLC1 could serve as a prognostic marker for colon cancer.

Discussion In the present study, we detected the expression of TSLC1 and then explored the clinical prognostic value of TSLC1 by using complete long-term follow-up data of a cohort of colon cancer samples. The results showed that TSLC1 was significantly down-regulated at both mRNA and protein levels in colon cancer tissues as compared with their corresponding normal tissues. Furthermore, tissue immunohistochemistry analysis indicated that decreased expression of TSLC1 was found in 41.3 % of the primary colon cancer tissues, and down-regulated TSLC1 expression was

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Fig. 2 Immunohistochemical staining of TSLC1 protein in colon cancer tissue samples. Tissue sections were immunohistochemically stained with an anti-TSLC1 antibody and scored as 0 (a), 1? (b), 2?

(c) and 3? (d). Positive TSLC1 immunostaining was mainly localized in the cytoplasm of colon cancer cells

significantly correlated with female gender, lymph node metastasis, and shorter overall survival of patients with colon cancer. Taken together, our data suggest that TSLC1 might represent a novel indicator for the prognosis of colon cancer. Mounting evidence has demonstrated that TSLC1, a novel tumor suppressor gene, is frequently inactivated by CpG island hypermethylation of its promoter region in a wide variety of human cancers, such as lung (Fukami et al. 2003), esophageal (Ito et al. 2003), nasopharyngeal (Hui et al. 2003), cervical (Steenbergen et al. 2004), breast (Heller et al. 2007), and colorectal cancer (Chen et al. 2011). In addition, loss or reduced expression of TSLC1 has also been reported to be correlated with poor patient survival in lung (Goto et al. 2005), esophageal (Ito et al. 2003), and ovarian cancer (Yang et al. 2011), neuroblastoma (Ando et al. 2008), and cutaneous melanoma (You et al. 2010). To the best of our knowledge, however, the prognostic significance of TSLC1 in colon cancer has not yet been investigated. In the present study, we detected the expression of TSLC1 in 30 paired colon cancer tissues by quantitative real-time PCR and Western blot analysis, and

then assessed TSLC1 protein expression in 80 primary colon cancer tissues by immunohistochemistry. The complete clinical follow-up data with various clinicopathologic parameters were available for review and correlation with TSLC1 immunohistochemistry analysis results. Our data showed that down-regulation of TSLC1 mRNA occurred in more than half of the colon cancer specimens (56.7 %, 17/30), and the relative level of TSLC1 mRNA expression in primary colon cancer tissues was significantly lower than that in corresponding non-tumor colon tissues, suggesting that down-regulation of TSLC1 may be an important event in the development and progression of colon cancer. In addition, reduced TSLC1 protein expression was found to be significantly correlated with female gender and lymph node metastasis of colon cancer patients. More importantly, consistent with previous results in other malignancies, we also found that decreased expression of TSLC1 in colon cancer was associated with shortened patient survival and was a reliable predictor for shortened overall survival by both univariate and multivariate analysis. Therefore, TSLC1 may serve as a novel prognostic marker for patients with colon cancer.

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Table 2 Associations between TSLC1 expression and clinicopathological factors of colon cancer patients Variables

Number of cases

TSLC1 protein expression High (n = 47)

Low (n = 33)

Male

45

22 (48.9 %)

23 (51.1 %)

Female

35

25 (71.4 %)

10 (28.6 %)

C65

43

25 (58.1 %)

18 (41.9 %)

\65

37

22 (59.5 %)

15 (40.5 %)

P value

Gender 0.042

Age (years) 0.905

Tumor differentiation Wellmoderate

63

38 (60.3 %)

25 (39.7 %)

Poor

17

9 (52.9 %)

8 (47.1 %)

52

31 (59.6 %)

21 (40.4 %)

28

16 (57.1 %)

12 (42.9 %)

Tumor size B5 cm [5 cm

0.584

0.830

TNM stage I/II

50

28 (56.0 %)

22 (44.0 %)

III/IV

30

19 (63.3 %)

11 (36.7 %)

0.519

Lymph node metastasis Negative

37

27 (73.0 %)

10 (27.0 %)

Positive

43

20 (46.5 %)

23 (53.5 %)

0.016

Previous studies have shown that promoter methylation is a key mechanism responsible for the loss or reduced expression of TSLC1 in various cancer types (Liang et al. 2011). Nonetheless, emerging evidence suggests that inactivation of TSLC1 may also occur via mechanisms other than promoter hypermethylation in certain cancers. For instance, Ando et al. reported that hypermethylation of TSLC1 promoter region is not detected in primary neuroblastomas and neuroblastoma-derived cell lines with loss or decreased expression of TSLC1 (Ando et al. 2008). Additionally, TSLC1 has been recently identified as a direct target of Polycomb protein Bmi1 in neuroblastoma Table 3 Univariate and multivariate analysis of prognostic factors in 80 colon cancer patients

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Variables

Fig. 3 Kaplan-Meier analysis of overall survival rate of colon cancer patients with high TSLC1 expression (n = 47) and low TSLC1 expression (n = 33), respectively. The overall survival rate between the two groups showed significantly different (P = 0.037, log-rank test)

cells. Bmi1 is a target gene of MYCN. Thus, these findings suggest that MYCN induces the expression of Bmi1, which in turn leads to the repression of TSLC1 through Polycomb group gene-mediated epigenetic chromosome modification (Ochiai et al. 2010). Taken together, despite Chen and coworkers have reported that promoter hypermethylation is a major contributor to the inactivation of TSLC1 in CRC, further studies are still required to precisely understand the molecular mechanism of TSLC1 inactivation in the development and progression of colon cancer and other human tumors. Furthermore, TSLC1 is a cell adhesion molecule expressed on the plasma membrane of cells in a wide range of normal tissues. However, in the present study, we found that immunoreactivity of TSLC1 protein was observed primarily in the cytoplasm of tumor cells in colon cancer tissues, which is consistent with previous reports in lung cancer (Goto et al. 2005) and cutaneous

Categories

Univariate analysis

Multivariate analysis

HR (95 % CI)

P value

HR (95 % CI)

2.38 (0.84–6.76)

0.103

–

Age

\65/C65

Gender

Male/female

0.71 (0.26–1.93)

0.504

Tumor differentiation

Well-moderate/ poor

1.74 (0.61–4.95)

0.296

Tumor size

B5 cm/[5 cm

1.47 (0.56–3.88)

0.433

TNM stage

I-II/III-IV

2.60 (0.99–6.85)

0.050

Lymph node metastasis

Negative/positive

2.17 (0.82–5.70)

0.117

TSLC1 protein expression

Low/high

2.75 (1.01–7.44)

0.016

P value

1.79 (0.65–4.92)

0.261

2.91 (1.22–10.41)

0.020

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melanoma (You et al. 2010). The molecular mechanism that is involved in this translocation remains unknown and needs to be elucidated. In summary, the present study demonstrated that TSLC1 is significantly decreased in surgically excised primary colon cancer tissues, and reduced TSLC1 expression correlates with female gender and lymph node metastasis and predicts poor prognosis in colon cancer patients. Our results indicate that TSLC1 appears to be an independent predictor of survival for patients with colon cancer.

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