Downregulating SynCAM and MPP6 expression is associated with ovarian cancer progression.

Synaptic cell adhesion molecules (SynCAMs) are single transmembrane proteins that belong to the immunoglobulin superfamily of cell adhesion molecules. In the present study, a decrease in SynCAM levels in ovarian tumor tissues compared with normal tissues is reported; the downregulation was accompanied by the grade malignancy. The observations suggested that SynCAM may be essential for important novel functions in ovarian cancer. Further experiments showed that low SynCAM expression inhibited membrane palmitoylated protein 6 (MPP6) expression, a member of the palmitoylated membrane protein subfamily of peripheral membrane-associated guanylate kinases. In addition, low levels of MPP6 in ovarian tumor tissues correlated with shorter patient survival. A SynCAM-regulated pathway may provide molecular targets for the treatment of ovarian cancer and novel biomarkers to be used in clinical diagnosis.

Introduction

Ovarian cancer (OC) is the most lethal gynecological malignancy (1). Surgical resection remains the primary option for patients with OC (2). High mortality rates (5-year survival rate, ~30%) of OC are based on late diagnosis, distant metastasis within 5 years after surgical treatment and chemotherapeutic resistance (3,4). Patients with different pathological stages of OC remain a considerable prognostic challenge (5). In a clinical setting, same stage tumors can lead to different outcomes (6). While mucin 16, cell surface associated and WAP four-disulfide core domain 2 levels exhibited great potential in the detection of high-grade serous ovarian carcinoma at later stages, the levels are not sensitive enough for early stage detection of this disease (7). Consequently, further understanding of the molecular mechanisms associated with the progression of OC is required.

Various cell adhesion molecules are located at synapses, but only few are considered synaptic cell adhesion molecules (8). Synaptic cell adhesion molecules (SynCAMs/CADMs) are a subfamily of the immunoglobulin superfamily of cell adhesion molecules (8). SynCAMs are single transmembrane proteins that were discovered in the central nervous system, due to their ability to induce synapse formation (9,10). SynCAMs are true synaptic cell adhesion molecules and are crucial for synapse formation and plasticity (11,12). SynCAM in the cytoplasmic domain contains the binding motifs that connect to actin fibers (8). SynCAMs are involved in synapse formation, neuronal connectivity, myelination and cerebellum morphogenesis (13–15). Furthermore, it was suggested that SynCAMs may contribute to autism spectrum disorder (16), glioma generation, non-small cell lung cancer and hepatocarcinogenesis (17,18). However, the prognostic importance of SynCAM expression and the associated underlying mechanisms has not fully been elucidated.

A biomarker can be defined as any measurable characteristic that provides an indication of the biological state of a patient (19). In the present study, SynCAM expression was investigated in 74 patients with OC using immunohistochemistry. In addition, membrane palmitoylated protein 6 (MPP6), a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK), levels were investigated in OC cells.

Materials and methods

Patients and tissue samples

The inclusion criteria were as follows: i) Patients agreed to surgical resection or chemotherapy; and ii) the all ovarian tumor was primary. The exclusion criteria were as follows: i) Patients that had refused any surgical resection or chemotherapy; ii) ovarian metastatic tumor; and iii) there was no sufficient tissue specimen for the immunohistochemical and western blot analyses. A total of 74 OC tissue specimens were derived from patients in The First Hospital of Lanzhou University (Lanzhou, China) between January 2011 and December 2012. None of the patients with OC had received chemo or radiotherapy prior to surgery. All patients underwent radical resection and were regularly followed up. The median age was 53 years (range, 17–75 years). The median follow-up time was 33 months (range, 6–60 months). The study was approved by the Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China) and written informed consent was obtained from the patients or their families. Borderline ovarian tumors (BOT; n=24) and benign ovarian tumor tissues (BEOT; n=34) were obtained from patients with ovarian tumors. Parts of the tumor specimens were frozen in liquid nitrogen after collection and stored at −80°C until use and other parts were formalin-fixed for 1 week at room temperature and paraffin-embedded.

Immunohistochemical staining

The specimens were cut into 4 µm sections. The deparaffinized specimens were washed twice with distilled water, 5 min at a time. Citrate buffer (pH 6.0) was used in antigen retrieval at 121°C for 20 min. After retrieval, the tissue sections were cooled for 20 min at room temperature. Finally, 0.01 mol/l PBS buffer was used to wash the sections twice for 5 min each time, and distilled water was then used to wash the sections three times for 3 min each time. To block endogenous peroxidase activity, 3% hydrogen peroxide was used for 15 min at 37°C. The slides were blocked with 10% normal goat serum (OriGene Technologies, Inc.) in PBS for 30 min at room temperature, and further incubated with rabbit anti-human primary monoclonal antibodies against SynCAM (1:500; cat. no. GR3184359-5; Abcam) and MPP6 (1:500; cat. no. 5324; Signalway Antibody LLC) at 4°C overnight. The following day the slides were incubated with ultraView universal HRP Multimer secondary antibody (1:1,000; cat. no. TA130015; anti-rabbit IgG Detection System; OriGene Technologies, Inc.) for 30 min at 37°C to assess protein expression, according to the manufacturer's protocol. Hematoxylin was used to stain cell nuclei for 1–2 min at 37°C. PBS was used as a negative control.

The tissue sections were assessed under a light microscope (Olympus Corporation) at ×10 magnification using the Allred scoring system (20). Brown-yellow staining was considered as positive protein expression. For the semi-quantitative evaluation of protein levels in the tissues, an immunoreactivity-scoring system was used as previously described (21). The staining intensity was graded (0, no stain; 1+, weak stain; 2+, moderate stain; and 3+, strong stain). High expression of SynCAM and MPP6 was defined as detectable immunoreactions in cytoplasm and membranes with scores ≥2.

Western blot analysis

Ovarian tumor tissues were homogenized in cold NP40 buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40; Beyotime Institute of Biotechnology), sonicated (20 KHz, 300 W, 5 min) on ice and lysates were centrifuged at 10,000 × g for 15 min at 4°C to collect the supernatant. The protein concentration was determined using a bicinchoninic acid assay (Beijing Solarbio Science & Technology Co., Ltd.). A total of 30 µg of protein per lane were separated on 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (OriGene Technologies, Inc.). Membranes were incubated with primary antibodies for SynCAM (dilution, 1:1,000; cat. no. GR3184359-5; Abcam), MPP6 (dilution, 1:1,000; cat. no. 5324; Signalway Antibody LLC) and β-actin (dilution, 1:5,000; cat. no. 14395-1-AP; Proteintech Group, Inc.) for 24 h at 4°C following a blocking step with 5% fat-free milk in TBST for 2 h at room temperature. Membranes were then incubated with secondary goat anti-rabbit IgG H&L antibody (1:1,000; cat. no. TA100015; OriGene Technologies, Inc.) for 1 h at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). Densitometry of the bands was performed using Quantity One software version 4.5.5 (Bio-Rad Laboratories, Inc.).

Statistical analysis

All data are presented as the mean ± standard deviation. Student's t-test was used for statistical analysis between two groups. P<0.05 considered to indicate a statistically significant difference. Positive expression rates and clinicopathological factors in the low and high SynCAM expression patients were compared using a χ2 test with a threshold value of P<0.05. Survival curves were plotted using the Kaplan-Meier method and were compared using the log-rank test. One-way ANOVA followed by Tukey post-hoc test were used to analysis of variance between groups. All statistical analyses were performed using the SPSS statistical software package (version 19.0; IBM, Corp.).

Results

Clinicopathological characteristics

Demographic, clinical and histopathological variables are presented in Table I. The current study included 74 patients with OC. The median age was 53 years (range, 17–75 years) and the cohort comprised 16 (21.62%) cases diagnosed at T1 and 12 (16.22%), 40 (54.05%) and 6 (8.11%) cases diagnosed at stages T2, T3 and T4 (TNM staging system) (22), respectively. The median follow-up time was 33 months (range, 6–60 months).

Table I.

Association between SynCAM expression and clinicopathological characteristics in patients with ovarian cancer.

		SynCAM expression (n=74)
Variables	Number (%)	Low expression	High expression	P-value
Age (years)				0.7886
<60	48 (64.86)	28	20
≥60	26 (35.14)	16	10
Tumor size (cm)				0.0308[a]
<8	50 (67.60)	34	16
≥8	24 (32.40)	10	14
T stage				0.1772
T1	16 (21.62)	10	6
T2	12 (16.22)	10	2
T3	40 (54.05)	22	18
T4	6 (8.11)	2	4
N stage				0.2509
N0	50 (67.57)	32	18
N1	24 (32.43)	12	12
M stage				0.5639
M0	66 (89.19)	40	26
M1	8 (10.81)	4	4
Differentiation level of tumor cells				<0.0001[a]
Low	18 (24.32)	2	16
High	56 (75.68)	42	14

P<0.01. SynCAM, synaptic cell adhesion molecules; T, tumor; N, node; M, metastasis.

Association between SynCAM expression and clinicopathological characteristics

In the present study, immunohistochemical analysis of 132 human ovarian tumor specimens was used evaluate SynCAM protein expression. SynCAMs exhibited cytolymph or cytoplasmic expression in ovarian tumor specimen. Representative images demonstrating SynCAM expression by staining BEOT, BOT and OC specimen were indicated (Fig. 1). Increased SynCAM expression was detected in BEOT (Fig. 1A) and BOT (Fig. 1B) compared with OC tissues (Fig. 1C). A total of 28/34 BEOT specimens exhibited positive SynCAM expression, representing a positive expression rate of 82.35% and 19/24 BOT cases exhibited positive SynCAM expression, representing a positive expression rate of 79.17%. Out of 74 OC specimen only 30 cases exhibited positive SynCAM expression, representing a positive expression rate of 40.54%. The majority of OC tissues exhibited no evidence for SynCAM staining. The positive expression rate was significantly increased in BEOT and BOT compared with OC (P<0.0001; Fig. 1D). The results suggested that SynCAM downregulation occurs in human ovarian tumor tissues. To further investigate the expression pattern of SynCAM in ovarian tumor tissues, SynCAM expression was examined by western blot analysis. The results showed that SynCAM levels were significantly decreased in OC and BOT compared with BEOT (P<0.001; Fig. 1E). Results demonstrated that SynCAM expression was decreased in all ovarian tumor tissues compared with normal tissues (Fig. 1E). The data suggested that SynCAM may function as a tumor suppressor in ovarian tumor tissues. SynCAM expression and the clinicopathological characteristics are presented in Table I. SynCAM expression was correlated with the tumor diameter (P=0.0308) and differentiation level of tumor cells (P<0.0001).

Figure 1.

SynCAM expression in ovarian tumor tissues. Representative images of SynCAM expression in (A) BEOT, (B) BOT and (C) OC specimen using immunohistochemical analysis (magnification, ×200 and ×400 for inset images). SynCAM exhibited cytoplasmic expression. (D) Positive expression rates of SynCAM were significantly decreased in OC compared with BEOT and BOT specimen. (E) The protein expression level of SynCAM was highest in BEOT specimens and significantly decreased in BOT and OC specimen; MPP6 expression was significantly downregulated in OC compared with BEOT and BOT and accompanied by the grade malignancy. *P<0.05, **P<0.01 and ***P<0.001. BEOT, benign ovarian tumor; BOT, borderline ovarian tumors; OC, ovarian cancer; SynCAM, synaptic cell adhesion molecules; MPP6, membrane palmitoylated protein 6.

MPP6 protein expression is downregulated in OC

Immunohistochemical analysis was used to evaluate MPP6 protein expression in ovarian tumor tissues. The MPP family belongs to the MAGUK family. MPP6 exhibited cytomembrane and cytoplasm expression in ovarian tumor tissues (Fig. 2). Fig. 2A-C are representative microphotographs of immunostained tissue sections of BEOT, BOT and OC, respectively, highlighting MPP6 staining. MPP6 expression in ovarian tumor samples was further determined by western blot analysis. The results suggested that MPP6 expression was significantly downregulated in OC compared with BEOT and BOT specimen (P<0.01 and P<0.05, respectively; Fig. 1E).

Figure 2.

MPP6 expression in ovarian tumor tissues. MPP6 expression in ovarian tumor specimens of (A) BEOT, (B) BOT and (C) OC (magnification, ×200 and ×400 for inset images). BEOT, benign ovarian tumor; BOT, borderline ovarian tumors; OC, ovarian cancer; MPP6, membrane palmitoylated protein 6.

Association between survival and expression of SynCAM and MPP6

SynCAM expression was not associated with overall survival (OS; n=74; P=0.8213; Fig. 3A). MPP6 expression was associated with poorer OS time; however not significantly (n=74; P=0.0934; Fig. 3B).

Figure 3.

Overall survival time curves of patients with ovarian cancer. Kaplan-Meier analysis according to (A) SynCAM and (B) MPP6 expression. SynCAM, synaptic cell adhesion molecules; MPP6, membrane palmitoylated protein 6.

Discussion

Metastasis, recurrence and chemotherapeutic resistance occur in patients with OC following radical resection are leading causes of mortality worldwide (23,24). SynCAMs, members of the immunoglobulin superfamily, encode for membrane glycoproteins and participate in cell adhesion (25). SynCAMs associate with different intracellular binding partners, including proteins of the MAGUK family (26,27). SynCAMs act as tumor suppressors in various types of human cancer including lung, prostate, pancreas and breast cancer, and are preferentially inactivated in invasive cancer (28,29). At present, to the best of our knowledge, little is known about the functional role of SynCAMs in OC. This study investigated the potential role of SynCAMs as tumor suppressors in OC.

SynCAMs are tumor suppressors in non-small cell lung cancer, hepatic cell carcinoma and pancreatic cancer (30,31). To the best of our knowledge, SynCAM expression in OC has not yet been reported. In the present study, it was demonstrated that SynCAM expression was downregulated in human OC tumor tissues compared with normal tissues, using immunohistochemical and western blot analyses. The majority of paraffin-embedded human OC tissues exhibited no evidence for SynCAM staining. The loss of SynCAM expression was correlated with increased tumor size and differentiation levels of tumor cells. SynCAM was expressed at higher levels in normal ovarian tissue compared with OC tissue. These findings demonstrated that SynCAMs may serve a tumor suppressive role in human OC. It is suggested that OC cell proliferation may be affected by downregulating SynCAM expression. Loss of SynCAMs expression is observed in non-small cell lung cancer, and could cause morphological transformation leading cancer cells to invasion and/or metastasis (28). The results of the present study are consistent with this finding.

Downregulation of SynCAM expression may further be associated with silenced methylation (32). SynCAM mRNA levels were described to be increased after demethylation in glioblastoma cell lines (32). However, the mechanism of SynCAM inactivation by promoter hypermethylation remains to be elucidated. MPP6, a member of the palmitoylated membrane protein subfamily of peripheral MAGUK, is primarily involved in controlling epithelial cell polarity (33). MPPs further function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal and cytoplasmic signaling proteins (34). A direct association between tumor suppression and cell polarity proteins is equivocal in mammals (35). Disruption of cell polarity and function causes abnormalities in vertebrates (35). Similar to E-cadherin, MPP loss can disturb cell-cell junctions and the mechanical integrity of epithelial cells, resulting in defective branching morphogenesis and maintenance of epithelial tubular structures (36–38). Saa3 is a member of the acute-phase serum amyloid A (SAA) apolipoprotein family. Murine Saa3 has been shown to be expressed in macrophages (39) and adipose tissue (40). Accumulation of SAA proteins in the blood is observed during chronic inflammation and cancer (39). Saa3 expression is effectively induced by interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 through NF-κB signaling in inflammatory processes (41). Inflammatory diseases greatly increase the risk of cancer, due to elevated expression of inflammatory cytokines, including IL-6, TNF, and IL-1β (42). Stimulating IL-23 and IL-17 production can promote cancer development and progression (42). MPPs can interact with SynCAM and form a protein complex (28). The protumorigenic activity of Saa3 can be regulated by MPP6. MPP6 overexpression is responsible for the loss of the protumorigenic effect of Saa3 in cancer-associated fibroblasts (41) (Fig. 4). In the present study, MPP6 immunolocalization was examined in OC tissues. MPP6 exhibited cytomembrane and cytoplasm staining and expression was significantly downregulated in ovarian tumor tissues compared with normal tissues. SynCAM and MPP6 expression may be used as prognostic factors to predict OS for individual patients with OC. However, SynCAM was determined not to be a prognostic factor and MPP6 expression was not significantly associated with worse OS, which may be due to the small sample size of OC in the present study. Further experiments are necessary to elucidate the potential association between SynCAM and MPP6.

Figure 4.

Schematic representation of the mechanism of SynCAM and MPP6 expression downregulation and its association with ovarian cancer progression. SynCAM, synaptic cell adhesion molecules; MPP6, membrane palmitoylated protein 6.

In summary, the data suggested that human OC cells proliferate through downregulation of SynCAM expression. It is suggested that decreased expression of SynCAM was associated with downregulation of the tight junction protein MPP6. Therefore, a SynCAM-regulated pathway may be an important molecular target in the treatment of ovarian cancer and the associated biomarkers may be used in a diagnostic clinical setting.