Sage Journals: Discover world-class research

Abstract

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the expression of PCDH8 and its clinicopathological significance in hypopharyngeal carcinoma. We also examined the possible inactivation mechanism of PCDH8. A total of 80 pairs of hypopharyngeal carcinoma tumor tissues and non-tumor tissues were investigated to examine the immunohistochemical expression of PCDH8. Prognostic value and clinicopathological significance of PCDH8 expression were examined by Kaplan-Meier and log-rank test and Cox’s ones. Ten pairs of tumor tissues and non-tumor tissues were analyzed by RT-PCR and 4 pairs by Western blot respectively. Promoter methylation of PCDH8 was observed in 14 pairs of tumor tissues and non-tumor tissues by methylation-specific PCR (MSP). The expression of PCDH8 in tumor tissues was depressed immunohistochemically when compared with non-tumor tissues and was significantly lower in the advanced pathological stage. Meanwhile, the expression of PCDH8 served as an independent prognostic risk factor for overall survival of hypopharyngeal carcinoma. The mRNA and protein levels of PCDH8 in tumor tissues was also down-regulated than in non-tumor tissues. Moreover, aberrant promoter methylation of PCDH8 occurred frequently in tumor tissues, rather than in non-tumor tissues. For the first time, our study demonstrates the tumor-suppressive function of PCDH8 and its epigenetic inactivation by promoter methylation in hypopharyngeal carcinoma. It suggests that PCDH8 may serve as a useful prognostic biomarker and a potential therapeutic target for hypopharyngeal carcinoma patients.

Keywords

Hypopharyngeal carcinoma protocadherin8 tumor suppressor gene

1. Introduction

Hypopharynx, also known as laryngopharynx, located behind the throat and on both sides, starting from below the hyoid extension line, ends at the lower edge of the annular cartilage connected to the esophagus. The incidence of hypopharyngeal carcinoma lacks detailed statistical data. Hypopharyngeal carcinoma accounts for about 5% of head and neck cancer, and is predominantly squamous cell carcinoma [19]. Hypopharyngeal carcinoma includes primary hypopharyngeal therioma and adjacent advanced cancer invasion [21]. Because of the special anatomical structure, the pathogenesis of hypopharyngeal carcinoma is concealed, and the exposure of Hypopharynx is difficult. Due to the late occurrence of clinical symptoms, the clinical pathological stage is often advanced (stages III and IV), meanwhile, about 50% of patients accompany with cervical lymph node metastasis. Therefore, the prognosis of hypopharyngeal carcinoma is generally poor [3]. Although advancements have been achieved in treating head and neck cancer with adjuvant chemotherapy, radiotherapy, and targeted molecular therapies, surgical intervention is still the main clinical treatment. According to recent studies, the 5-year survival rate of radiotherapy alone is 12.7% $\sim$ 13.9%, and the radical surgery and postoperative radiotherapy 5-year survival rate is 25% $\sim$ 60% [13, 14, 17]. Hence, it is of great important to identify novel prognostic and predictive markers to evaluate the occurrence, progression, and prognosis of the disease.

The protocadherins (PCDHs), a subfamily of the cadherin superfamily, contain 6 extracellular cadherin domains, a transmembrane domain, and different cytoplasmic domains [5, 22]. The PCDHs play a mediating role as regulators of other molecules and cell-cell adhesion [22]. As a member of the PCDHs, PCDH8 is reported to play a significant role in cell adhesion, signal transduction, proliferation, migration, and invasion in nasopharyngeal carcinoma [1], breast cancer [9], renal cell carcinoma [15], gastric cancer [2] and bladder cancer [27]. Meanwhile, the expression of PCDH8 is frequently depressed in these human cancers. However, whether this gene is subject to down-regulated expression in hypopharyngeal carcinoma remains unclear.

As is well-known that epigenetic changes are frequently involved in tumor development and progression, including DNA promoter CPG island (CGI) methylation and histone modifications [16]. Therefore, identification of epigenetic changes involved in the initiation and progression of hypopharyngeal carcinoma may identify novel diagnostic and epigenetic biomarkers and therapeutic targets for early detection of hypopharyngeal carcinoma. According to series of studies, epigenetic mechanism plays a crucial role in tumor development and progression. A numbers of genes, such as Zinc-Finger Protein 545 [25], Jagged1 [28], LATS2 [29] and ZNF382 [26], have already been reported to be frequently silenced by the methylation of DNA promoter, serving as tumor suppressors. However, such epigenetic changes of PCDH8 have not been reported yet in hypopharyngeal carcinoma.

In the present study, we verified the depressed expression and promoter methylation of PCDH8 in isolated hypopharyngeal carcinoma tissues of surgical patients. We also evaluated the relationship between the expression of PCDH8 and clinicopathological features of hypopharyngeal carcinoma. Moreover, we examined the correlation between PCDH8 expression and the patients’ overall survival. On the basis of the research, we found that PCDH8 may be a functional tumor suppressor in the development and progression of hypopharyngeal carcinoma. Furthermore, PCDH8 may serve as a prognostic biomarker for hypopharyngeal carcinoma patients.

2. Materials and methods

2.1 Patients and tissue samples

A total of 80 pairs of tumor tissues and adjacent non-tumor tissues were obtained from 80 patients with hypopharyngeal carcinoma during laryngopharyngectomy at the First Affiliated Hospital of Chongqing Medical University between 2012 and 2017. Each pair of tumor tissue and adjacent tissue was obtained from isolated hypopharyngeal carcinoma tissues resected from the surgery. These tissues were examined by experienced pathologists according to the World Health Organization classification to exclude the possibility of incidental tumors. Informed consent was obtained from each participant before any treatment. Significantly, none of the patients with hypopharyngeal carcinoma received any form of anti-tumor therapy or adjuvant chemotherapy before surgery. This study was approved by the ethics committee of Chongqing Medical University. All tissue samples were flash-frozen in liquid nitrogen at the time of collection and then stored at $-$ 80 ${}^{\circ}$ C until used. Tumor tissues were used for the experiment group and non-tumor tissues were used for control group. Meanwhile, the common clinicopathologic features of patients with hypopharyngeal carcinoma were recorded.

2.2 Immunohistochemistry

Immunohistochemistry for PCDH8 was performed on 4 $\mu$ m sections of formalin fixed, paraffin embedded tissues including 80 pairs of tumor tissues and adjacent non-tumor tissues mentioned above. Following deparaffinization in xylene and rehydration in graded alcohol, antigen retrieval was performed with citrate buffer, pH 6.0 at 100 ${{}^{\circ}}$ C for 30 minutes. Slides were pretreated with Peroxidase Block (ZSGB-BIO, Beijing, China) for 15 minutes to ensure that endogenous peroxidase activity was quenched, and then rinsed in Phosphate buffer saline (PBS) three times for 3 minutes each. Primary PCDH8 antibody (Abcam, Cambridge, UK) was applied at 1:100 dilution, and slides were incubated in antibody diluent at 37 ${{}^{\circ}}$ C for 2 hours. Slides were then rinsed in Phosphate buffer saline (PBS) three times for 3 minutes each and treated with boost IHC detection reagent (ZSGB-BIO, Beijing, China) for 20 minutes at 37 ${{}^{\circ}}$ C. After further rinse, staining development was achieved by incubation with 3, 3-diaminobenzidine (DAB) and DAB Enhancer (ZSGB-BIO, Beijing, China) for 5 minutes. After all above steps, slides were counterstained with hematoxylin for about 30 seconds, dehydrated in xylene and graded alcohol, and then coverslipped.

The immunohistochemistry results were analyzed by two different experienced pathologists and scored using a double-blind method. Five high-power fields ( $\times$ 400) were selected at random, and two pathologists evaluated scores independently. Slides without primary antibody incubation were selected as negative controls, and slides with PCDH8 antibody (Abcam, Cambridge, UK) used on formalin fixed and paraffin embedded human tonsil were selected as positive controls. According to the intensity and total area of the staining, the expression of PCDH8 was scored as either positive expression (expressed $\geqslant$ 50% of cells) or negative expression ( $<$ 50% of cells).

2.3 Total-RNA extraction and RT-PCR

According to the instructions for the Trizol total RNA extraction kit (TaKaRa, Japan), total RNA was extracted from 10 pairs of tumor tissues and adjacent non-tumor tissues of patients with hypopharyngeal carcinoma mentioned above. The harvested RNA was reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan) and then stored at $-$ 80 ${{}^{\circ}}$ C until use. GAPDH served as an internal reference for RNA integrity. The primers used were: PCDH8-F: 5’-CGACAGCGATTCCGACATCA -3’ (Sangon Biotech, Shanghai, China), PCDH8-R: 5’-CTCTGCAGCCCCACTGTCTT-3’ (Sangon Biotech, Shanghai, China), GAPDH-F: 5’-GGATGACCTTGC CCACAGGGT-3’ (Sangon Biotech, Shanghai, China), GAPDH-R: 50-GTTGGGGGTTCTGGGGACTGGC-3’ (Sangon Biotech, Shanghai, China). PCR was performed for 50 cycles with anannealing temperature of 55 ${{}^{\circ}}$ C by CFX Connect Optics Module Real-Time System (BIO-RAD, Hercules, CA, USA).

2.4 Protein extraction and Western blotting analysis

Proteins were extracted from tumor tissues and adjacent non-tumor tissues using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), followed by centrifugation at 12000 $\times$ g for 15 minutes at 4 ${{}^{\circ}}$ C. The protein lysates were then loaded into 10% sodium dodecyl sulphate-polyacryla- mide gels, and transferred onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China). Membranes were blocked with 5% skimmed milk for 1 hour, and incubated with primary antibodies overnight at 4 ${{}^{\circ}}$ C and then with secondary antibodies for 2 hours in room temperature. Primary PCDH8 antibody (Abcam, Cambridge, UK) was applied at 1:1000 dilution, primary antibody against $\beta$ -actin (Biodragon, Beijing, China) was applied at 1:1000 dilution using for internal reference, and secondary antibody against mouse or rabbit IgG (BOSTER, Wuhan, China) was applied at 1:2000 dilution. The protein band signals were detected with an ECL Kit (Wanleibio, Shenyang, China) and visualized by ChemiDoc Touch Imaging System (BIO-RAD, Hercules, CA, USA).

2.5 DNA extraction and methylation-specific PCR (MSP)

Genomic DNA was extracted from tumor tissues and adjacent non-tumor tissues using GenElute Mammalian Genomic DNA Miniprep Kit (SIGMA- ALORICH, St. Louis, MO, USA). The integrity of DNA was detected by gel electrophoresis. Samples were stored at $-$ 80 ${{}^{\circ}}$ C until use. Bisulfite modification of DNA and methylation-specific PCR(MSP) were performed as described in representative studies [18]. Bisulfite-treated DNA was amplified by MSP with the methylation-specific primer set PCDH8-m1: 5’-C GGTTATTGGTTATTCGGTTCC-3’, PCDH8-m2: 5’- ACGAACTCTAAAAACGCGCG-3’, or the unme- thylation-specific primer set PCDH8-u1: 5’-GGTGGT TATTGGTTATTTGGTTT-3’, PCDH8-u2: 5’-CCAAC AAACTCTAAAAACACACA-3’. MSP was performed for 40 cycles using AmpliTaq-Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA) with an annealing temperature of 60 ${{}^{\circ}}$ C for methylation-specific primer and an annealing temperature of 58 ${{}^{\circ}}$ C for unmethylation-specific primer. Methylation-spe- cific PCR (MSP) primers have been tested to be specific, amplifying no bisulfited genomic DNA sample.

Figure 1.

(A) Positive immunohistochemistry for PCDH8. The numbers of PCDH8 positive cells were more than 50%. (B) Negative immunohistochemistry for PCDH8. The numbers of PCDH8 positive cells were less than 50%. (C) Slide without primary antibody incubation was selected as negative control. The magnification of all photomicrographs was $\times$ 400. (D) Slide with PCDH8 antibody used on human tonsil was selected as positive control.

2.6 Statistical analysis

Statistical analyses were performed with SPSS version 22.0 software (SPSS Inc, Chicago, IL, USA). The associations between the expression of PCDH8 and clinicopathological features were evaluated by chi-square test. For overall survival analysis, Kaplan-Meier survival analysis was used and the differences in survival were analyzed using the log-rank test. Univariate and multivariate Cox proportional hazard models were used to evaluate the prognostic effect of PCDH8 expression in hypopharyngeal carcinoma. The difference of PCDH8 methylation status between tumor tissues and non-tumor tissues were evaluated using Fisher’s exact test. Results for the RT-PCR assays were presented as mean $\pm$ SD. For all tests, $p$ -value $<$ 0.05 was considered statistical significance.

3. Results

3.1 Identification of PCDH8 deletion in immunohistochemistry

According to the Immunohistochemistry score method described previous, the expression of PCDH8 in 80 tumor tissues and 80 adjacent non-tumor tissues was evaluated to be positive or negative (Fig. 1). More importantly, we found that the expression of PCDH8 was significantly down-regulated in tumor tissues (60/80, 75.0%) when compared to the adjacent non-tumor tissues (18/80, 22.5%) ( $P<$ 0.001) (Table 1).

Table 1
The immunohistochemical expression of PCDH8 in tumor tissues and non-tumor tissues

Tissue	Number of	Expression of PCDH8		$P$ Value ${}^{\#}$
	patients	Positive	Negative
Tumor	80	20	60	$<$ 0.001
Non-tumor	80	62	18

${}^{\#}P$ values are from $\chi^{2}$ test or Fisher’s exact test and were statistically significant when $<$ 0.05.

Table 2

Correlations between expression of PCDH8 and clinicopathological features of hypopharyngeal carcinoma patients

Feature	Clinicopathologic parameters	Number of patients	Expression of PCDH8		$P$ Value ${}^{\#}$
			Positive	Negative
Sex	Male	78	20	58	1.000
	Female	2	0	2
Age(years)	$<$ 60	33	7	26	0.512
	$\geqslant$ 60	47	13	34
Smoking	Yes	68	16	52	0.718
	No	12	4	8
Pathological stage	T1–T2	14	7	7	0.041
	T3–T4	66	13	53
Lymph node metastasis	Presence	62	12	50	0.064
	Absence	18	8	10

${}^{\#}P$ values are from $\chi^{2}$ test or Fisher’s exact test and were statistically significant when $<$ 0.05.

Table 3

Univariate and multivariate Cox regression analyses of overall survival in hypopharyngeal carcinoma patients

Variable	Univariate analyses			Multivariate analyses
	HR	95% CI	$P^{\#}$	HR	95% CI	$P^{\#}$
Age	3.043	1.548–6.601	0.0343	2.373	1.192–4.725	0.0139
Smoking	1.332	0.962–4.279	0.5421
Pathological stage	1.144	0.788–5.474	0.9720
Lymph node metastasis	1.825	1.056–6.749	0.7328
Expression of PCDH8	3.791	1.543–10.566	0.0260	3.704	1.306–10.509	0.0138

${}^{\#}P$ values are from log-rank test or Cox analyses were statistically significant when $<$ 0.05.

3.2 Clinicopathological significance and prognostic value of PCDH8 expression

Statistical analyses of the correlation between the expression of PCDH8 and clinicopathological parameters are summarized in Table 2. Obviously, the expression of PCDH8 was down-regulated in advanced pathological stage (T3–T4) compared to early pathological stage (T1–T2) ( $P=$ 0.041). However, no significant differences were observed between expression of PCDH8 with regard to age, sex, smoking, or the lymph node metastasis (Table 2). Kaplan-Meier survival analysis and log-rank test results suggested that patients with PCDH8 positive expression had significantly longer overall survival than patients with PCDH8 negative expression (Fig. 2, $P=$ 0.0184). This result indicates that PCDH8 expression was significantly associated with prognosis of patients with hypopharyngeal carcinoma. Univariate Cox regression analysis first showed that age and the expression of PCDH8 were risk factors for poor overall survival. These risk factors were entered into multivariate Cox regression analysis, revealing that age and PCDH8 expression were independent prognostic risk factors for overall survival of patients with hypopharyngeal carcinoma. These results are summarized in Table 3.

Figure 2.

Correlations between the expression of PCDH8 and overall survival in patients with hypopharyngeal carcinoma. Patients with PCDH8 positive expression showed significant longer overall survival than those with PCDH8 negative expression (log-rank test, $P=$ 0.0184, chi-square $=$ 5.5567).

3.3 Reduced expression of PCDH8 in RT-PCR and Western blotting analysis

To investigate whether PCDH8 was aberrantly expressed in hypopharyngeal carcinoma, we first examined PCDH8 mRNA level in 10 pairs of hypopharyngeal carcinoma tumor tissues and adjacent non-tumor tissues using real-time PCR (RT-PCR). We found that PCDH8 expression was significantly downregulated in 100% (10/10) of hypopharyngeal carcinoma tumor tissues when compared to the adjacent non-tumor tissues ( $P=$ 0.0002). Similar trend was also observed at the protein level by Western blotting analysis in 4 pairs of tumor tissues and non-tumor tissues (Fig. 3).

Figure 3.

The expression level of PCDH20 in tumor tissues and non-tumor tissues was measured by quantitative RT-PCR and Western blot assay, respectively. (a) Expression of PCDH8 mRNA level by real-time RT-PCR. The abscissa indicates the type of tissues. The ordinate indicates the relative mRNA expression levels of PCDH8. The line in the grouped column scatter represented mean values of PCDH8 mRNA ( $P<$ 0.05). (b) Expression of PCDH8 protein level by Western blot assay, $\beta$ -actin was served as loading control. N: non-tumor tissue; T: tumor tissue.

3.4 Methylation of the protocadherin8 promoter contributes to its downregulation in hypopharyngeal carcinoma

In the current study, the methylation status of PCDH8 in 14 pairs of hypopharyngeal carcinoma tumor tissues and non-tumor tissues was detected by methylation-specific PCR(MSP). PCDH8 methylation was detected in 7 (50.0%) tumor tissues compared to 1 non-tumor tissue (7.1%) (Fig. 4). Meanwhile, the difference between tumor tissues and non-tumor tissues was statistically significant ( $P=$ 0.033) (Table 4).

Table 4
The Methylation of PCDH8 in tumor tissues and non-tumor tissues

Tissue	Number of	Methylation of PCDH8		$P$ Value ${}^{\#}$
	patients	Methylated	Unmethylated
Tumor	14	7	7	0.033
Non-tumor	14	1	13

${}^{\#}P$ values are from $\chi^{2}$ test or Fisher’s exact test and were statistically significant when $<$ 0.05.

Figure 4.

Representative analysis of methylation of the PCDH8 promoter in hypopharyngeal carcinoma tumor tissues and adjacent non-tumor tissues. Methylation-specific polymerase chain reaction (MSP) results are shown. M: methylated; U: unmethylated; N: non-tumor tissue; T: tumor tissue.

4. Discussion

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common and aggressive head and neck cancers with poor prognosis and high recurrence [8, 24]. Although advances have been made in surgery, radiotherapy and chemotherapy, the 5-year survival rate of patients with HSCC ranges from 40 to 50%, with a morbidity of 1 case per 100,000 people [6, 12, 23]. It is of great important to identify novel prognostic and predictive markers to understand this multifaceted disease process and to identify which patients need more aggressive treatment after initial curative surgery. Fortunately, PCDH8 has been reported as an effective tumor suppressor involved in tumorigenesis in cancers [1, 2, 9, 15, 27], according to a variety of clinical studies and cytology experiments. Meanwhile, the decreased expression of PCDH8 has also been verified in these cancers above, contributing to its down-regulated tumor-suppressive function. However, whether the expression of PCDH8 is reduced in hypopharyngeal carcinoma and whether this gene can be a potential tumor biomarker and therapeutic target in hypopharyngeal carcinoma remain unclear.

The protocadherins (PCDHs), different from classic cadherins, are not just cell-adhesion proteins involved in homophilic interactions, and their heterophilic interactions with other molecules may be more significant for their various physiological functions [5, 22]. DNA methylation of CpG islands within the promoter region of genes is an alternative mechanism of gene silence to genetic changes and is frequently involved in the development and progression of many types of human cancers. Aberrant promoter methylation of some genes that are normally unmethylated may be used as potential molecular markers for the diagnosis, surveillance, and prognosis in hypopharyngeal carcinoma. As for several members of PCDHs, expression silence or functional inactivation, caused by mutations or promoter methylation, for example, PCDH8 [1, 2, 9, 15, 27], PCDH10 [4, 10, 11], PCDH17 [20], and PCDH20 [7], has been discovered in varies of cancers, resulting in tumor cell growth, invasion, and metastasis. These results are complementary to our data and also hint that epigenetic inactivation of PCDHs in tumors could be widespread indeed. Therefore, Protocadherin8 (PCDH8) may have multiple tumor-suppressive functions, such as the involvement in cell-cell adhesion, signal transduction, and growth control. However, the association between the function of PCDH8 and the prognosis of hypopharyngeal carcinoma have not been reported yet. This is the first study to investigate the prognostic value of PCDH8 expression and methylation in hypopharyngeal carcinoma based on a relatively large number of clinical samples.

In the current study, we found that PCDH8, localized on human chromosome 13q14.3, was frequently downregulated or totally silenced in 75% hypopharyngeal carcinoma tumor tissues (60/80), but widely expressed in non-tumor tissues (62/80) according to Immunohistochemistry. More importantly, we found that depressed PCDH8 expression was significantly correlated with advanced pathologic stage. Meanwhile, PCDH8 was proved to serve as an independent prognostic risk factor for overall survival of hypopharyngeal carcinoma patients according to Kaplan-Meier & log-rank test and Cox’s ones. Whether it implicates an early diagnosis of carcinogenesis should be an interesting question. Further, we found that PCDH8 functions as a tumor suppressor by RT-PCR and Western blotting analysis. Our data clearly demonstrated that mRNA level and protein expression of PCDH8 is frequently decreased in tumor tissues compared to paired non-tumor tissues. These results suggest that PCDH8 plays an important role in the progression of hypopharyngeal carcinoma. Moreover, methylation-specific PCR (MSP) demonstrated that aberrant promoter methylation of PCDH8 occurred frequently in tumor tissues, rather than in non-tumor tissues. This result indicates that PCDH8 methylation is tumor-specific and may be an early event in development and progression of hypopharyngeal carcinoma.

In summary, we demonstrated in this study that PCDH8 can inhibit the development and progression of hypopharyngeal carcinoma, and examined the clinicopathological significance and prognostic value of PCDH8 expression. We also verified the frequent promoter methylation in hypopharyngeal carcinoma in a tumor specific manner as a possible epigenetic inactivation mechanism. Our data strongly support the notion that PCDH8 is a candidate tumor suppressor in hypopharyngeal carcinoma and may serve as a useful prognostic biomarker and a potential therapeutic target for hypopharyngeal carcinoma patients. It would thus be worthwhile to conduct further study to investigate the cause of PCDH8 methylation and to discover how to reverse the methylation status of PCDH8 in human hypopharyngeal tissues. Meanwhile, the functional mechanism of tumor suppressor also remains further intensive study.

Footnotes

Acknowledgments

This study was supported by the Natural Science Foundation of Key Projects of Chongqing China (Grant Number. cstc2012jjB10015). All authors thank Department of Biochemistry and Molecular Biology, Molecular Medicine and Cancer Research, China Center, Chongqing Medical University for technical assistance and encouragement. The authors contributed equally to this work.

Conflict of interest

The authors declare no conflict of interest.

References

Zeng

Ren

et al., Protocadherin8 is a functional tumor suppressor frequently inactivated by promoter methylation in nasopharyngeal carcinoma, European Journal of Cancer Prevention the Official Journal of the European Cancer Prevention Organisation 21 (2012), 569.

Zhang

Zhao

Liao

et al., Frequent silencing of protocadherin 8 by promoter methylation, a candidate tumor suppressor for human gastric cancer, Oncology Reports 28 (2012), 1785–1791.

Mura

Bertino

Occhini

and Benazzo

, Surgical treatment of hypopharyngeal cancer: a review of the literature and proposal for a decisional flow-chart, Acta Otorhinolaryngol Ital 33 (2013), 299–306.

Narayan

Scotto

Neelakantan

et al., Protocadherin PCDH10, involved in tumor progression, is a frequent and early target of promoter hypermethylation in cervical cancer, Genes Chromosomes Cancer 48 (2010), 983–992.

Morishita

and Yagi

, Protocadherin family: diversity, structure, and function, Current Opinion in Cell Biology 19 (2007), 584–592.

Hoffman

H.T.

Karnell

L.H.

Shah

J.P.

et al., Hypopharyngeal Cancer Patient Care Evaluation, Laryngoscope 107 (2010), 1005–1017.

Imoto

Izumi

Yokoi

et al., Frequent silencing of the candidate tumor suppressor PCDH20 by epigenetic mechanism in non-small-cell lung cancers, Cancer Research 66 (2006), 4617–4626.

Spector

J.G.

Sessions

D.G.

Haughey

B.H.

et al., Delayed regional metastases, distant metastases, and second primary malignancies in squamous cell carcinomas of the larynx and hypopharynx, Laryngoscope 111 (2010), 1079–1087.

J.S.

Koujak

Nagase

et al., PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer, Oncogene 27 (2008), 4657–4665.

10.

Tao

Cheung

K.F.

et al., Epigenetic identification of ubiquitin carboxyl-terminal hydrolase L1 as a functional tumor suppressor and biomarker for hepatocellular carcinoma and other digestive tumors, Hepatology 48 (2010), 508–518.

11.

Cheng

Y.Y.

Tao

et al., Methylation of protocadherin 10, a novel tumor suppressor, is associated with poor prognosis in patients with gastric cancer, Gastroenterology 136 (2009), 640–651.

12.

Davies

and Welch

H.G.

, Epidemiology of head and neck cancer in the United States, Otolaryngol Head Neck Surg 135 (2006), 451–457.

13.

Lambert

Fortin

Soulières

et al., Organ preservation with concurrent chemoradiation for advanced laryngeal cancer: are we succeeding? Int J Radiat Oncol Biol Phys 76 (2010), 398–402.

14.

Alexios Martin

M.D.

Jäckel

M.C.

Hans Christiansen

M.D.

et al., Organ Preserving Transoral Laser Microsurgery for Cancer of the Hypopharynx…, Laryngoscope 118 (2010), 398–402.

15.

Morris

M.R.

Ricketts

C.J.

Gentle

et al., Genome-wide methylation analysis identifies epigenetically inactivated candidate tumour suppressor genes in renal cell carcinoma, Oncogene 30 (2011), 1390.

16.

Jones

P.A.

and Baylin

S.B.

, The Epigenomics of Cancer, Cell 128 (2007), 683–692.

17.

Chu

P.Y.

and Chang

S.Y.

, Reconstruction of the hypopharynx after surgical treatment of squamous cell carcinoma, Journal of the Chinese Medical Association 72 (2009), 351–355.

18.

Tao

Huang

Geiman

T.M.

et al., Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells, Human Molecular Genetics 11 (2002), 2091.

19.

Patel

R.S.

Goldstein

D.P.

Brown

et al., Circumferential pharyngeal reconstruction: History, critical analysis of techniques, and current therapeutic recommendations, Head & Neck 32 (2010), 109–120.

20.

Haruki

Imoto

I.K.

Matsui

et al., Frequent silencing of protocadherin 17, a candidate tumour suppressor for esophageal squamous cell carcinoma, Carcinogenesis 31 (2010), 1027.

21.

Wiegand

Esters

Müller

H.H.

et al., Is it necessary to dissect levels I and IIB in hypopharyngeal cancer? Acta Otolaryngol 130 (2010), 747–752.

22.

Kim

S.Y.

Yasuda

Tanaka

et al., Non-clustered protocadherin, Cell Adhesion & Migration 5 (2011), 97–105.

23.

Gupta

Chopra

Agarwal

J.P.

et al., Squamous cell carcinoma of the hypopharynx: single-institution outcome analysis of a large cohort of patients treated with primary non-surgical approaches, Acta Oncologica 48 (2009), 541–548.

24.

Helliwell

T.R.

, Best Practice No 169: Evidence based pathology: squamous carcinoma of the hypopharynx, Journal of Clinical Pathology 56 (2003), 81–85.

25.

Yun

Xiang

Luo

et al., Zinc-Finger Protein 545 Inhibits Cell Proliferation as a Tumor Suppressor through Inducing Apoptosis and is Disrupted by Promoter Methylation in Breast Cancer, Plos One 9 (2014), e110990.

26.

Cheng

Geng

Cheng

S.H.

et al., KRAB zinc finger protein ZNF382 is a proapoptotic tumor suppressor that represses multiple oncogenes and is commonly silenced in multiple carcinomas, Cancer Research 70 (2010), 6516–6526.

27.

Lin

Y.L.

Wang

Y.L.

J.G.

and Li

W.P.

, Clinical significance of protocadherin 8 (PCDH8) promoter methylation in non-muscle invasive bladder cancer, Journal of Experimental & Clinical Cancer Research 33 (2014), 1–6.

28.

Cao

Y.W.

Y.X.

Zhang

et al., Quantitative DNA hypomethylation of ligand Jagged1 and receptor Notch1 signifies occurrence and progression of breast carcinoma, Am J Cancer Res 5 (2015), 1621–1634.

29.

Yan

C.F.

Jing

et al., LATS2 is De-methylated and Overexpressed in Nasopharyngeal Carcinoma and Predicts Poor Prognosis, Bmc Cancer 10 (2010), 1–13.

Expression of protocadherin8: Function as a tumor suppressor in hypopharyngeal carcinoma

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and tissue samples

2.2 Immunohistochemistry

2.3 Total-RNA extraction and RT-PCR

2.4 Protein extraction and Western blotting analysis

2.5 DNA extraction and methylation-specific PCR (MSP)

3. Results

3.1 Identification of PCDH8 deletion in immunohistochemistry

Table 1 The immunohistochemical expression of PCDH8 in tumor tissues and non-tumor tissues

Table 4 The Methylation of PCDH8 in tumor tissues and non-tumor tissues

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
The immunohistochemical expression of PCDH8 in tumor tissues and non-tumor tissues

Table 4
The Methylation of PCDH8 in tumor tissues and non-tumor tissues