Membrane Proteins as Potential Colon Cancer Biomarkers : Verification of 4 Candidates from a Secretome Dataset

Colorectal cancer (CRC) is an important health issue in Taiwan. There were over ten thousand newly diagnosed CRC patients each year. The outcome of late stage CRC still remains to be improved, and tumor markers are expected to improve CRC detection and management. From a colorectal cancer cell secretome database, we chose four proteins as candidates for clinical verification, including tumor-associated calcium signal transducer 2 (TROP2, TACSTD2), transmembrane 9 superfamily member 2 (TM9SF2), and tetraspanin-6 (TSPAN6), and tumor necrosis factor receptor superfamily member 16 (NGFR). Different groups of 30 CRC patients’ tissue samples collected from Chang Gung Memorial Hospital were analyzed by immunohistochemistry (IHC) for the four proteins, and the results were scored by pathologist. For all the four candidate proteins, marked differences of IHC score existed between tumor and adjacent non-tumor counterpart. However, there were only trends between higher protein expression levels and worse outcome. Three proteins (TROP2, TM9SF2 and NGFR) had trends between higher tissue expression and tumor stage or lymph node metastasis. Our study revealed that tissue expression of four proteins (TROP2, TM9SF2, TSPAN6, and NGFR) was markedly different between tumor and adjacent non-tumor counterparts. Overexpression of all these four proteins showed some trends with poorer survival. Corresponding authors.


Introduction
In Taiwan, colorectal cancer (CRC) has become an important health issue in recent years.There were more and more newly diagnosed CRC every year.In 2011, there were 14,087 CRC patients diagnosed in Taiwan [1].Among them, 49.5% were diagnosed as late stage (stage III, 26.6%; stage IV, 22.8%).The five-year survival rate for late staged CRC was still relatively low (for colon cancer, stage III, 47.8%; stage IV, 10.3%) [2].Tumor biomarkers were expected to integrate into CRC management to improve earlier diagnosis rate, to improve risk stratification, and to predict treatment response [3]- [7].However, carcinoembryonic antigen (CEA), the most common serum biomarker currently used in clinic for CRC, doesn't meet all these clinical needs [8]- [12].
Although serum biomarkers play important role in cancer management, histology-based approaches are still the gold standard for tumor staging at present.Molecular staging provides an opportunity to personalized medicine and to maximize cost-effective management [13]- [16].More and more analyses suggest that molecular approaches offer an advantage for stratifying patients further.Recently, some prospective studies have begun to predict risk of disease recurrence by molecular markers [17] [18].For example, epidermal growth factor receptor (EGFR), which participates in signaling pathways that are deregulated in cancer cells and up-regulated in 50% -80% CRC cases [19]- [24], has shown to be related to prognosis and therapeutic response of CRC patients [25] [26].Many studies reported that Cetuximab, an antibody against EGFR, has additional benefit on CRC patients receiving chemotherapy [27] [28].
In recent years, the secretome-based approach has been proven to be a promising strategy for discovery of CRC biomarkers [29]- [31].Using this approach, we have previously established a secretome dataset from 23 cancer cell lines, from which over one hundred proteins were selected identified from two CRC cell lines [29].Among them, we had selected four protein candidates for verification, including tumor-associated calcium signal transducer 2 (TROP2, TACSTD2), transmembrane 9 superfamily member 2 (TM9SF2), tetraspanin-6 (TSP-AN6), and tumor necrosis factor receptor superfamily member 16 (NGFR).These four proteins have been preliminarily examined in some CRC tissues in the Human Protein Atlas (HPA).Furthermore, there have been some reports about expression levels of TROP2 in different cancers including pancreatic cancer, CRC and ovarian carcinoma [32]- [35].Tumor necrosis factor receptor superfamily member 16 (NGFR) has been studied in neurologic malignancy, which is also involved in cell growth control [36]- [39].In functional aspect, TSPAN6 was found to be involved in cell motility [40] [41].which may be related to tumor cell migration.We verified these four proteins in CRC tissues and their adjacent non-tumor counterparts by immunohistochemistry.We also analyzed the relationships between protein expression levels and clinicopathological factors of CRC patients.

Patient Population and Clinical Specimen
All clinical samples were collected at Chang Gung Memorial Hospital (Taoyuan, Taiwan).Tissue samples were collected in 1995.Different groups of 30 CRC patients' tissue samples were used for our candidates.All CRC patients had histologically verified adenocarcinoma.All were subjected to a follow-up strategy that included regular outpatient visits, CEA test, and image studies.Patients characteristics, including gender, age, tumor location, histological grade, tumor stage, CEA level, operation date, tumor recurrence, follow up date, and follow up status, were obtained from clinical and pathology records.The study was approved by the Institutional Review Board at Chang Gung Memorial Hospital (IRB No. 99-0515B, 101-0712B and 102-1446C).

Immunohistochemistry
The tumor tissue blocks used for IHC were first fixed in 4% paraformaldehyde and then embedded in paraffin.
Sections (5 μm thick) were cut from tissue blocks, mounted on silanized slides (Superfrost, Menzel, Braunschweig, Germany), subsequently deparaffinized with xylene (twice for 10 min each), and rehydrated through ethanol gradient washes.Endogenous peroxidase activities are inactivated in 3% H 2 O 2 before heating in a microwave oven for antigen retrieval (10 mm citrate buffer, pH 6.0; 20 min, 700 W).To block nonspecific binding, slides were preincubated with 10% nonimmune goat serum at 37˚C for 30 min.Slides were then incubated with anti-human primary antibody for 30 min at room temperature.Following washing with PBS (pH 7.4), slides were incubated with HRP-conjugated IgG secondary antibody for 30 min at room temperature and then developed using 3,3'-diaminobenzidine (Sigma, St Louis, MO).All procedure followed the standard pro-tocol.Expression of these protein was categorized as positive or negative and was evaluated according to the percentage of cells stained (0% -100%) and the intensity of cell staining (3, strong; 2, moderate; 1, weak; or 0, no cell staining).

Statistical Analysis
For the analysis of IHC results, independent t test was used.The associations between protein expression and clinicopathological characteristics were analyzed using chi-square method and ANOVA.To determine factors related to overall survival, the probability was calculated using the Log-rank test by the Kaplan-Meier method.Cox proportional hazard models were used for maltivariate analysis.Comparative analysis of IHC scoring and CEA in paired CRC patients were undertaken.Statistical significance was set at p < 0.05.All analyses were performed using the statistical software, Statistical Package for the Social Sciences (Version 17.0, SPSS Inc., Chicago, IL).

Clinicopathological Analysis between High and Low Protein Expression Groups
All patients of each studied group for four candidate were divided into high expression group and low expression group.We used medium IHC scoring as cut-off value of high and low expression.It were 100 for TROP2, 110 for TM9SF2, 150 for TSPAN6, 110 for NGFR.We analyzed most clinicopathologic factors, including ages, gender, tumor location, histological grade, tumor stage, T stage, N stage, CEA level, and survival.It seems no differences between high and low expression groups for each protein (Table 1).

Comparison of IHC Stained Area and Staining Scoring According to Different Clinicopathological Factors
Furthermore, we compared mean proteins expression area and IHC scoring between different clinicopathologic factors.Although most factors had no statistical differences, patients with worse 5-year survival had trends of higher proteins expression area and IHC scoring.Patients with late tumor stage or positive lymph node metastasis had trends of higher protein expression (for TROP2, TM9SF2, NGFR).The trends also existed in histological grade (for TROP2 and NGFR) (Table 2).

Kaplan-Meier Survival Analysis According to High and Low Protein Expression
For all four proteins, high expression groups has the trend of worse survival, especial at 10-year.For TROP2, the 10-year survival rate of high expression and low expression groups were 28.5% vs. 50.0%(p = 0.43).For TM9SF2, the 10-year survival rate of high expression and low expression groups were18.0%vs. 42.0%(p = 0.14).For TSPAN6, the 10-year survival rate of high expression and low expression groups were 22.0% vs. 33.0%(p = 0.60).For NGFR, the 10-year survival rate of high expression and low expression groups were 52.9% vs. 61.5% (p = 0.62) (Figure 2).

Multivariate Analysis
Using 5-year survival as end point, we further did multivariate analysis for all four proteins (Table 3).However, TROP2 and NGFR didn't show any differences.High TM9SF2 expression group had HR 1.22 (p = 0.72).High TSPAN6 expression group had HR 3.75 (p = 0.02).

Comparison Analysis with CEA
All four proteins tissue expression seemed not to be related to CEA level.However, all candidates increased detection of normal CEA cases.We used medium IHC scoring as cut off for each protein.They were 100 for TROP2, 110 for TM9SF2, 150 for TSPAN6, and 110 for NGFR, respectively.For TROP2, 6 among 16 normal CEA cases had higher TROP2 tissue expression.For TM9SF2, 6 among 15 normal CEA cases had higher tissue expression.For TSPAN6, 11 among 17 normal CEA cases had higher tissue expression.For NGFR, 7 among 14 normal CEA cases had higher tissue expression (Figure 3).All four proteins had the potential to improve false negative rate of CEA.

Expression Analysis among Stages with Normal and Abnormal CEA Levels
We further analyzed the percentage of high and low expression among early stage and late stage cases.We found that, for TROP2 and TM9SF2, late stage with abnormal CEA had high tissue protein expression, compared to early stage with normal.But it seemed not to be different for NGFR, and even to have reverse association for TSPAN6 (Figure 4).

Discussion
At present, more and more biomarkers were analyzed in clinical setting.EGFR, which participates in signaling pathways, has shown to be associated with treatment response [19] [20].These tumor biomarkers, especially on  tissue level, were expected to improve earlier diagnosis rate and to make management more individualized [5] [7] [15].
Our study verified four membrane proteins from a secretome dataset in tissue level.The IHC scores of four candidate proteins were markedly different between tumor and adjuvant normal.Although further analysis did not show statistical difference, all of these proteins showed some trends with poorer survival.Except TSPAN6, other three proteins (TROP2, TM9SF2, NGFR) showed some trends with tumor aggressiveness (tumor stage, lymph node metastasis).Statistical insignificance might be due to small sample sizes.
In the literature, several tissue biomarkers had been verified in CRC (Supplement Table 1).Most of them are associated with survival or prognosis (Supplement Table 1).At present, tissue markers, not serum markers, can be used as a predictor of treatment response.For example, tissue expression of EGFR, which had been verified as biomarker of treatment response [27], was also a biomarker of survival.Survivin, an inhibitor of apoptosis, is known to be expressed in most tumor cell types.Several studies had shown its potential as a target for cancer therapy [42] [43].Tissue biomarkers, which were tested more widely, seem to have the potential to be integrated in CRC management.Unlike serum markers, tissue markers research is more likely to be a straightforward strategy for treatment markers discovery.
Our data didn't usually show consistency between four candidates and different analyses.And p values were not significant because of small sample sizes.However, marked IHC scoring differences existed between tumor and adjuvant normal, and most candidates showed some trends with tumor aggressiveness and survival.In comparison analysis with CEA, our candidate proteins also showed the potential of improving false negative

Figure 3 .
Figure 3. Medium IHC scores were used as cut off values for each proteins.(a) For TROP2, 6 among 16 normal CEA cases had higher TROP2 tissue expression.(b) For TM9SF2, 6 among 15 normal CEA cases had higher tissue expression.(c) For TSPAN6, 11 among 17 normal CEA cases had higher tissue expression.(d) For NGFR, 7 among 14 normal CEA cases had higher tissue expression.

Figure 4 .
Figure 4. Comparison of the percentages of high and low protein expression.For TROP2 and TM9SF2, more percentages of high protein expression were found in late stage CRC patients with abnormal CEA.For TSPAN6 and NGFR, the associations were reverse.

Table 1 .
Analysis of clinicopathologic factors of different proteins expression groups.

Table 2 .
Comparison of protein IHC scaring according to different clinicopathologic factors.
NS: not significant.NA: not available.

Table 1 .
rate of CEA.More cases to be tested are needful.These four membrane proteins still have the potential to be novel CRC biomarkers.More studies are needed to integrate these proteins in clinical usage.The association between serum and tissue expression is the next interesting issue.Prioritization and selection of candidate biomarkers from CRC cell secretomes.