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Abstract

BACKGROUND:

Downregulation of PCDH20 is frequently involved in tumorigenesis of many cancers, but the role of PCDH20 protein in hypopharyngeal squamous cell carcinoma (HSCC) is still unknown.

OBJECTIVE:

The aim of this study was to investigate the role of PCDH20 in hypopharyngeal squamous cell carcinoma (HSCC).

METHODS:

Immunohistochemistry (IHC) and qRT-PCR was carried out to estimate the expressions of PCDH20 protein and mRNA in HSCC tissues and adjacent non-tumor tissues. Correlation between the PCDH20 expression and clinicopathological characteristics was evaluated using chi-square test. Meanwhile, Kaplan-Meier method and log-rank test were applied to analyze the overall survival. After transfection of PCDH20, the CCK8 assay, Cell migration assay and invasion assay were used to investigate the changes in the viability, migration and invasion of Fuda cells. The mechanisms by which reduced PCDH20 promote migration and invasion of Fuda cells were examined using western blotting.

RESULTS:

PCDH20 protein showed in tumor tissue low expression rates of 67.5% (54/80). The mRNA of PCDH20 indicated the consistent trend (80%, 8/10). Reduced PCDH20 expression was positively related to T stage and lymph node metastasis ( $P<$ 0.05). Patients with low levels of PCDH20 had worse overall survival compared with those with high PCDH20 levels ( $P<$ 0.001). The univariate Cox regression analysis described that lymph node metastasis ( $P=$ 0.043) and down-regulated PCDH20 expression ( $P=$ 0.045) were significantly prognostic factors.Multivariate analysis suggested that low PCDH20 expression ( $P=$ 0.015) were significantly independent prognostic factors for overall survival. PCDH20 in Fadu cells significantly inhibited cell viability, migration and invasion. Meanwhile, PCDH20 was involved in the disruption of HSCC progression through antagonizing its downstream Wnt/ $\beta$ -catenin signalling pathway.

CONCLUSION:

Our data highlight that the downregulated PCDH20 may serve as reliable diagnostic biomarker in HSCC.

Keywords

PCDH20 hypopharyngeal squamous cell carcinoma clinicopathological features lymph node metastasis prognostic

1. Introduction

Hypopharyngeal squamous cell carcinoma (HSCC), considered as one of the common malignant tumors of head and neck cancer, accounts for about 5% [19]. Owning to the anatomical structure and submucosal infiltration, the early clinical symptoms of the patients are not greatly apparent. Conseqently, when diagnosed, most of the patients are in the advanced stage or metastasis which is the main cause of treatment failure. Recent studies showed that the 5-year survival rate of patients is only 15–30% [12, 13, 16]. Hence, it’s quite urgent to explore the pathogenesis and expound special diagnostic biomarkers for HSCC.

Cadherins, acted as a key role in the cell-cell communication, which contains at least 80 members. Among these, more than 60 protoc adherins have been identified and are currently a major subfamily of the cadherin superfamily [15, 23]. The protocadherins (PCDHs), divided into two subtypes: “clustered” and “non-clustered” groups, is a kind of calcium-dependent molecule [5, 20]. Several members of the PCDHs family, such as PCDH8 in nasopharyngeal carcinoma [4], PCDH10 in gastric cancer and cervical cancer [27, 14], and PCDH17 in esophageal squamous cell carcinoma,gastric and colorectal cancers [3, 7], have recently been reported to be involved in the disruption of carcinogenesis. Regrettably, the relationship between PCDHs and the pathogenesis of the other cancers remains unclear. PCDH20, belonging to PCDHs family, contains 6 extracellular cadherin regions, a transmembrane region and a spore tail region [18]. Recent studies have shown that PCDH20, acted as tumor suppressor, frequently inhibited proliferation, repressed migration and invasion, blocked cell cycle, and promoted apoptosis in variety of cancers, such as non-small-cell lung cancers [8], nasopharyngeal carcinoma [21], and hepatocellular carcinoma [9]. Nevertheless, the relationship between PCDH20 expression and HSCC has never been reported in the previous studies.

In the present study, we clarified the expression of PCDH20 in HSCC tissues and non-tumor tissues. In addition, we estimated the correlation between the expression of PCDH20 and prognosis of HSCC. According to the above data, we further evaluated the biological function and molecular mechanism of PCDH20 in Fadu cells.

2. Materials and methods

2.1 Patients and specimens

Here in this study, a total of 80 patients with HSCC and corresponding non-tumor tissues were collected from the department of Head and Neck Surgery, First Affiliated Hospital of Chongqing Medical University, China, between 2007 and 2012. Absolutely, the specimens were diagnosed by biopsy at pathology department of Chongqing Medical University according to the admitted standard. All these samples were saved with snap-frozen in liquid nitrogen, then transmitted into $-$ 80 ${}^{\circ}$ C fridge for later use. Among these, 10 pairs of fresh HSCC and corresponding non-tumor samples were used for qRT-PCR. All surgical samples including 80 HSCC tissues and corresponding non-tumor tissues were fixed with 10% buffered formalin and embedded in paraffin. This protocol was approved by the Ethics Committee of Chongqing Medical University.

2.2 RNA extracting and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA of 10 coupled HSCC and matched non-tumor samples were extracted with Trizol reagent (Takala, Dalian, China) based on the manufacturer’s instruction. RNA intensity and purity ranged from 1.8 to 2.0 inspected by spectrophotometry. Briefly, the prepared RNA were reversely transcribed into cDNA using PrimeScript RT Reagent Kit (Takala, Dalian, China) according to the manufacturer’s specification. Then 0.5 $\mu$ g cDNA, as a template, was used for qRT-PCR with SYBR Premix Ex TaqTMII (Takala, Dalian, China) according to the manufacturer’s instructions. GAPDH was provided to normalize RNA integrity as a kind of housekeeping gene. The primers used were:

PCDH20-forward 5’-TCTACATCGTCCCAGGA GCA-3’ PCDH20-reverse 5’-GCGGGTAGTCCCTCGTTT AG-3’ GAPDH-forward 5’-GAAGGTGAAGGTCGGAG TC-3’ GAPDH-reverse 5’-GAAGATGGTGATGGGATT TC-3’

The qRT-PCR was carried out for 37 ${}^{\circ}$ C for 15 min, 85 ${}^{\circ}$ C for 5 sec, 4 ${}^{\circ}$ C for 1 min.

The relative expression levels of mRNA were calculated by 2 ${}^{-\Delta\Delta\text{CT}}$ method. All assays were performed in triplicate, and the mean value was used for the analysis.

2.3 Immunohistochemistry (IHC)

The expression of PCDH20 protein of 80 pairs HSCC patients and matched non-tumor tissues were tested by immunohistochemistry (IHC). Shortly, on the first day, histologic sections were dried at 60 ${}^{\circ}$ C overnight. After deparaffinized with xylene and rehydrated with the graded alcohol. the slides were boiled by citrate buffer (1 mmol/L, PH $=$ 6.0) for 20 min using microwave oven at 98 ${}^{\circ}$ C for antigen repair, then natural cooling about 40 min. Subsequently, the 3% H ${}_{2}$ O ${}_{2}$ was applied for blocking the endogenous peroxidase activity for 10 min. The sections were hatched with goat serum for 20 min. Finally, The sections were incubated with Rabbit anti-PCDH20 polyclonal antibody (1:40 dilution; sc-84558, Santa Cruz, USA) overnight at 4 ${}^{\circ}$ C. On the second day ,the slides were rewarmed at 37 ${}^{\circ}$ C for 1 h, then the HRP-conjugated of popular type, Mouse/Rabbit, secondary antibody (ZSGB-BIO, Beijing, China) were used to treat the slides at 37 ${}^{\circ}$ C for 30 min. After washing three times with phosphate buffer saline, the slides were hatched with the streptavidin-horseradish peroxidase complex. At last, the DAB (ZSGB-BIO, Beijing, China) was applied for visualizing the slides, followed by counterstaining with hematoxylin and viewed with an Olympus microscope. As negative control, the primary antibody was substituted for normal goat serum.

Figure 1.

The expression level of PCDH20 in tumor tissues and non-tumor tissues was measured by qRT-PCR and IHC assay, respectively. (A). Relative mRNA expression of PCDH20 in HSCC tissues displayed significantly lower than that in non-tumor tissues by qRT-PCR. Paired T-test was for the statistical analysis ( $p=$ 0.015). (B). IHC results showed HSCC tissues presented negative or weak immunohistochemical staining of PCDH20. (I) (original magnification, 400) whereas non-tumor tissues presented high staining (II) (original magnification, 400). I represented the low expression of PCDH20, whereas II represented high expression of PCDH20. ( $p<$ 0.05).

2.4 Cell culture and transfection

HSCC cell line Fuda was obtained from Cell Bank of Chinese Academy of Science (Shanghai, China). The cells were cultured at 37 ${}^{\circ}$ C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured overnight in a 6-well plate (3 $\times$ 10 ${}^{5}$ /well). Lipofectamine 2000 (Invitrogen, Carls bad, CA, USA) was used to transfect the PCDH20 expression plasmid and empty vector (0.6 mg each) into Fuda cells.Then, cells were hatched for 48 h. Total RNA from the transfected cells was extracted and analyzed by qRT-PCR to confirm the ectopic expression of PCDH20. Whereafter, relevant functional experiments were carried out.

2.5 Cell counting kit-8 (CCK-8) assay

Fadu cells were inoculated into 96-well plates at a density of 1 $\times$ 10 ${}^{3}$ cells/well. CCK-8 kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to detect cell viability. The optical density (OD) of each sample was measured at a wavelength of 450 nm on a microplate reader after 0 Day, 1 Day, 2 Day and 3 Day of transfection, and the growth curve was plotted.

2.6 Cell migration and invasion assay

After 48 h transfection, Fadu cells suspensions were prepared with serum-free medium. And the number of cells was counted by 1 $\times$ 10 ${}^{5}$ cells/mL. Cell migration and invasion was carried out using transwell chambers (24-well). The bottom chambers were filled with DMEM containing 10% FBS. The upper chambers were filled with serum-free medium. A total of 200 $\mu$ l of cell suspension was slowly added to the upper chamber of each Transwell chamber with or without Matrigel (for invasion and migration, respectively). After incubation for 24 h, 48 h, 72 h, non-invading cells in the upper of the membrane were removed with a cotton swab. The cells migrated to the bottom side of the membrane were fixed by methanol, stained with crystal violet for 30 min and counted.

2.7 Western blotting analysis

Lipofectamine 2000 was used to transfect the PCDH20 expression plasmid and empty vector into Fuda cells. Cell proteins were collected with a mixture of ice-cold lysis buffer (Applygen, Beijing, China) (RIPA) and 1% Phenylmethanesulfonyl fluoride (PMSF) (Beytime, Biotechnology, Shanghai, China) and examined with the standard BCA method (BCA ${}^{\rm TM}$ Protein Assay Kit, Pierce, USA). Then, loading buffer of 5 $\times$ sodium dodecyl sulfate (SDS) was added into the protein lysates, followed boiling at 100 ${}^{\circ}$ C for 10 min. Briefly, for Western blotting, the purified protein lysates were electrophoresed with SDS-PAGE gels, and PDVF membranes (Millipore, Billerica, MA, USA), used to be transferred, were blocked with 10% skimmed milk for 2 h. Subsequently, the membrane was washed with Tris-HCl buffer saline containing Tween-20 buffer three times, following incubation with secondary antibody for 2 h. The antibodies used were PCDH20, $\beta$ -catenin, GSK3 $\beta$ , anti-mouse IgG, anti-rabbit IgG and GAPDH. Finally, The protein bands were detected with imaging system.

2.8 Statistical analysis

The results were expressed as mean $\pm$ standard. All statistical analyses were performed for using the SPSS 19.0 statistical software package (SPSS Inc., Chicago, IL, USA). Comparisons between two groups were analyzed by independent-sample $t$ -test or paired $t$ -test.Correlation between the PCDH20 expression and clinicopathological characteristics was evaluated using chi-square test, Kaplan-Meier method and log-rank test were applied to examine the overall survival. Univariate and Multivariate analyses of prognostic factors were carried out using Cox proportional hazards regression model. $P<$ 0.05 was considered to be statistically significant.

3. Results

3.1 Downregulated PCDH20 expression in HSCC

The expression level of PCDH20 in 10 paired HSCC samples and corresponding non-tumor tissues was detected by qRT-PCR, normalized with a control (GAPDH). The results turned out that PCDH20 expression was significantly low in 80% (8/10) of HSCC tissues compared that in the non-tumor tissues ( $p<$ 0.05). Representative data were showed in Fig. 1.

3.2 Relationship between PCDH20 expression and clinicopathological characteristics

A total of 80 pairs of HSCC and corresponding adjacent non-tumor tissues were examined by IHC with special antibody. As shown in Table 1, of all 80 HSCC cases, 67.5% (54/80) showed reduced PCDH20 expression. Moreover, PCDH20 was frequently located in the membrane and cytoplasm of adjacent normal tissues (Fig. 1). The result showed that the negative rate of PCDH20 expression in HSCC was significantly higher than that in the non-tumor tissues (chi-text, $P<$ 0.05).

Table 1
Relationship between clinicopathological features and expression of PCDH20 protein

Variables	Cases	PCDH20 expression
		Positive	Negtive	$P$ value
Gender				1.000
Male	78	23	55
Female	2	0	2
Age(y)				0.207
$<$ 60 years	33	12	21
$\geqslant$ 60 years	47	11	36
Smoking				0.130
Yes	67	17	50
No	13	6	7
T stage				0.000*
T1–T2	20	14	6
T3-T4	60	9	51
Lymph node metastasis				0.001 ${}^{*}$
Presence	57	10	47
Absence	23	13	10
Tumor	80	26	54	0.000 ${}^{*}$
Non-tumor	80	59	21

$P<$ 0.05 represents statistically significant (chi-square test or Fisher’s exact test).

Since the expression of PCDH20 was abnormal in HSCC, we explored the role of PCDH20 during tumor progression. Thus, the relationship between PCDH20 expression and clinicopathological characteristics was estimated in our study. The results indicated that no significant differences were found between expression of PCDH20 with sex, age, smoking ( $P>$ 0.05), however, there were significantly associated with the advanced T stage ( $P<$ 0.001), lymph node metastasis ( $P=$ 0.001), as shown in Table 1.

3.3 Prognostic significance of PCDH20 expression in HSCC

To determine the prognostic value of PCDH20 expression in HSCC, Kaplan-Meier curves for overall survival are described in Fig. 2. The curves depicted that patients with low levels of PCDH20 had worse overall survival compared with those with high PCDH20 levels (log-rank $P<$ 0.001, Fig. 2). Moreover, the univariate Cox regression analysis described that Lymph node metastasis ( $P=$ 0.043) and down-regulated PCDH20 expression ( $P=$ 0.045) were significantly prognostic factors. Meanwhile, multivariate analysis suggested that lymph node metastasis ( $P=$ 0.012) and low PCDH20 expression ( $P=$ 0.015) were significantly independent prognostic factors for overall survival (Table 2).

Table 2
Univariate and multivariate Cox regression analysis of overall survival in 80 patients with HSCC

	Univariate analyses			Multivariate analyses
	$P$	RR	95.0% CI	$P$	RR	95.0% CI
Age
$<$ 60 versus $\geqslant$ 60	0.111	1.724	0.882–3.369
Smoking
Yes versus no	0.390	2.505	0.308–20.359
T stage
T1, T2 versus T3, T4	0.334	0.343	0.039–3.009
Lymph node metastasis
N+ versus N-	0.043 ${}^{*}$	8.002	1.069–60.178	0.012 ${}^{*}$	12.774	1.736–94.005
PCDH20
High versus low	0.045 ${}^{*}$	0.118	0.015–0.955	0.015 ${}^{*}$	0.083	0.011–0.614

RR risk ratio, CI confidence interval. $P<$ 0.05 represents statistically significant.

Figure 2.

The Kaplan-Meier survival curves constructed for HSCC patients with PCDH20 expression. Patients with low levels of PCDH20 had worse overall survival compared with those with high PCDH20 levels (log-rank $P<$ 0.001).

3.4 Loss of PCDH20 expression promoted viability, migration and invasion in Fadu cells

In the beginning, the expression of PCDH20 in Fadu cells was evaluated after transfected PCDH20 gene. As shown in Fig. 3A, the result demonstrated that the expression of PCDH20 was significantly higher in Fadu cells than that in vector cells ( $P<$ 0.05). Then, CCK8 assay was carried out to detect cell viability in Fadu cells. The result indicated that the survival rates of vector cells survival rates elevated significantly compared to the Fadu-PCDH20 cells ( $P<$ 0.05; Fig. 3B). Eventually, we examined the influences of PCDH20 on the migration and invasion of FaDu cells by Transwell and Matrigel assays, respectively. Results displayed that loss of PCDH20 expression in Fuda cells remarkably enhanced its migration ability ( $P<$ 0.05; Fig. 3C); the Matrigel showed the consistent trend ( $P<$ 0.05; Fig. 3D). These data suggested that loss of PCDH20 expression may promoted the oncogenesis and metastasis in HSCC.

Figure 3.

PCDH20 regulated cell viability, migration and invasion in Fadu cells. (A). The level of PCDH20 was significantly overexpressed after transfected PCDH20 gene in Fadu cells by qRT-PCR. (B). CCK-8 assays showed that PCDH20 inhibited cell viability in Fadu cells. (C). Migration assays showed that PCDH20 repressed the migration capacity of Fadu cells. (D). Invasion assays demonstrated that PCDH20 blocked the invasion of PCDH20 cells.* means $P<$ 0.05.

3.5 Subdued PCDH20 expression promoted Wnt/

\beta

-catenin signalling pathway activation

Recent studies reported that several members of PCDHs,such as PCDH10, PCDH17, could antagonize the Wnt/ $\beta$ -catenin signalling pathway [24, 26]. Hence, we explored the influence of PCDH20 on the signalling pathway in Fuda cells. As shown in Fig. 4A and B, subdued PCDH20 expression notably facilitated $\beta$ -catenin protein level compared to transfected PCDH20 gene according to western blotting assay ( $P<$ 0.05).

Figure 4.

PCDH20 regulated Wnt/ $\beta$ -catenin signaling pathway. (A, B) PCDH20 was negative correlation with Wnt/ $\beta$ -catenin signaling pathway using Western blotting. GAPDH was used as a loading control. * means $P<$ 0.05.

4. Discussion

As is known to all, hypopharyngeal squamous cell carcinoma (HSCC) is one of the common head and neck malignant cancers with increasing rates of morbidity and mortality [10, 22]. Despite of advanced treatment to reverse this condition, the 5-year survival rate for patients with HSCC remains no ideal, due to recurrence and metastases [6]. Hence, it is highly urgent to identify novel biomarkers to illuminate this allround disease process and to exploit the mechanism.

The protocadherin (PCDH) family, a kind of tumor suppressor gene family, is relatively well investigated in various human cancers. Several members of PCDHs, have been discovered in varieties of cancers, such as PCDH8 [4], PCDH10 [27, 14], PCDH17 [3, 7], inhibiting tumor cell growth, invasion, and metastasis. PCDH20, also known as PCDH13, which belongs to the subgroup of non-clustered protocadherin gene family, plays a crucial role not only in growth control but also in cell-cell communication. As a novel protocadher, PCDH20 is expressed predominantly in nervous system [2, 11]. Till now, there are limited studies with respect to the expression of PCDH20 in HSCC and its relationships with clinical characteristics and clinical outcome.

In the present study, Our results definitely showed that mRNA level of PCDH20 was frequently reduced in tumor tissues 80% (8/10) compared to paired non-tumor tissues by qRT-PCR. Meanwhile, the immunohistochemical results displayed that PCDH20 protein was notably downregulated in HSCC tissues 67.5% (54/80), which is concordant with previous reports. Imoto et al. [8] discovered PCDH20 was silenced in 54.2% non-small cell lung cancer (NSCLS) tissues. A decreased expression of PCDH20 in nasopharyngeal carcinoma but not in normal nasopharygeal tissues was observed [21]. All these results suggest that reduced PCDH20 expression is significantly associated with tumor progression. Therefore, we hypothesize that PCDH20 might participate in the development of HSCC.

A study on hepatocellular carcinoma (HCC) suggested that the reduced PCDH20 expression was associated with poor tumor differentiation [25]. In our study, tumors with negtive PCDH20 protein expression were more frequently observed in advanced T stage, lymph node metastasisin in HSCC. These results again demonstrated that downregulated PCDH20 might be involved in promoting the poor progression of HSCC. Meanwhile, it was showed in our study the PCDH20 expression was unrelated to age, which was not accordance with previous study [9], we suggested that the role of PCDH20 on age in HSCC need to be confirmed in more cases and further study was needed.

In HCC [25], NSCLS [8], decreased expression of PCDH20 was related to poor prognosis of these patients. Furthermore, PCDH20 could be used to be an independent prognostic factor for HCC [25]. Our group found that there was a positive correlation between PCDH20 expression and prognosis of HSCC. Cox regression analysis suggested PCDH20 could be an independent prognostic factor for patients with HSCC. Meanwhile, the Kaplan-Meier curves indicated that patients with low PCDH20 expression was significantly short overall survival compared to those with high PCDH20 expression. Therefore, we suppose analysis of PCDH20 expression might provide a useful additional prognostic tool in case of HSCC.

Further, we found that restoration of PCDH20 expression induced inhibition of Fadu cells proliferation, migration and invasion. The same observations that ectopic expression of PCDH20 inhibited cell migration and invasion are also identified in NPC cells [21]. Meanwhile, in other protocadherins, various tumor suppression mechanisms have been reported: PCDH10 inhibits cell proliferation via downregulation of Wnt/ $\beta$ -catenin/BCL-9 signaling [24]. PCDH17 promotes metastasis and invasion through hyperactivation of EGFR/MEK/ERK signaling pathway [1]. As is known, carcinogenesis is a multi-step process involving the accumulation of multiple genetic mediated by oncogenes.

Wnt signalling pathway plays an important role in the embryonic development of animals, and abnormal alterations of Wnt signalling pathway are the most common events associated with human carcinogenesis [17]. Recent studies reported that certain members of PCDHs can regulate the expression of some downstream targets of Wnt signalling pathway [24, 26, 28]. In this study, we are the first to demonstrate that PCDH20 suppresses the Wnt/ $\beta$ -catenin signalling pathway in Fadu cell lines. Further studies should be conducted to determine which intracellular molecule(s) mediating the inactivation induced by PCDH20.

In summary, we firstly validated that reduced PCDH20 expression can effectively promote the tumorigenesis of HSCC, and verified the relationship between clinicopathological features and PCDH20 expression. What’s more, we also futher demonstrated that PCDH20 can inhibit cell viability, migration and invasion through blocking Wnt/ $\beta$ -catenin signalling pathway in Fadu cells. Our results provide an opportunity for future target-guided therapy it might be participated in the development of HSCC and be utilized as a useful prognostic biomarker. Further studies are needed to validate these findings and to clarify the exact mechanism of PCDH20 loss of function in tumorigenesis.

Footnotes

Acknowledgments

This work was financed from the funds of the National Natural Science Foundation of China (81470676 and 81271061).

Conflict of interest

None.

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