Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma

Abstract

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51–6.75 (p = 0.002); 2.24, 1.06–4.76 (p = 0.035); and 2.56, 1.09–6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Keywords

VEGFR1/FLT1 VEGFR2/KDR VEGFR3/FLT4 head and neck squamous cell carcinoma methylation

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent type of cancer, affecting ~500,000 people per year worldwide.¹ Major risk factors for HNSCC include gender, tobacco smoking, alcohol consumption and human papillomavirus (HPV) infection.² The prognosis for patients with HNSCC remains poor despite tremendous advances in radiotherapy, chemotherapy and surgical techniques.³ Lymph node metastasis significantly worsens HNSCC prognosis;⁴ thus, identification of biomarkers that predict lymph node metastasis in HNSCC patients is imperative.

Vascular endothelial growth factor (VEGF) is often expressed in malignant tumours. It is a risk factor for metastasis and an indicator of poor prognosis in several cancer types including HNSCC.^5,6 It promotes angiogenesis for tumour growth and lymphangiogenesis for metastasis by binding to VEGF receptors (VEGFRs), of which there are three main subtypes (VEGFR1–3).^7,8 Numerous agents that target VEGF or VEGFRs, and thus prevent their interaction, have been developed for cancer therapy and widely tested;^9,10 these include antibodies, aptamers, peptides and small molecules.^11,12 Some of these agents have been approved by the Food and Drug Administration, and some are currently in clinical trials.

VEGFR expression has been linked to metastasis and poor prognosis^13–15 and antithetically to good prognosis.^16,17 In HNSCC, overexpression of VEGFR1 and VEGFR3 correlates with poor prognosis, tumour recurrence and lymph node metastasis, whereas overexpression of VEGFR2 correlates with the initiation of vasculogenesis and invasiveness.^18,19 VEGFR1 overexpression in the absence of lymph node metastasis has been observed, namely, in neck dissections of patients receiving curative surgery.^20,21

Epigenetic changes such as DNA methylation can predict lymph node metastasis and cancer prognosis.^22,23 Next-generation sequencing technology and recent advances in CpG methylation arrays have made it possible to detect cancer-related gene methylation comprehensively. Use of these techniques led to the identification of VEGFR methylation as an appropriate marker for early detection of colorectal cancer, oesophageal squamous cell carcinoma (SCC) and HNSCC.^24–26 However, methylation analysis of the individual VEGFR genes, notably VEGFR1, VEGFR2 and VEGFR3, in HNSCC requires further study.

The purpose of this study was twofold: to determine the methylation profiles of the VEGFR gene promoters in HNSCCs at the time of diagnosis and to evaluate the usefulness of VEGFR methylation as a prognostic indicator of tumour recurrence and patient survival. All three VEGFR genes were examined, as was the relationship between VEGFR methylation and various clinical characteristics. We show that methylation of the VEGFR promoters indicates poor prognosis in HNSCC.

Materials and methods

Cell lines and tumour samples

The HNSCC cell lines UM-SCC-6 (derived from the base of the tongue) and UM-SCC-47 (HPV-infected, derived from the lateral tongue) were obtained from the University of Michigan (Ann Arbor, MI, USA). The SCC cell line FaDu was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Waco, Osaka, Japan) (UM-SCC-6 and UM-SCC-47) or Dulbecco’s modified Eagle’s medium (Waco) (FaDu) supplemented with 10% foetal bovine serum (Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Waco) in a humidified atmosphere containing 5% CO₂ at 37°C.

All clinical specimens were surgically obtained from primary HNSCCs (n = 128) at Hamamatsu University School of Medicine. The samples were obtained soon after diagnosis and thus from untreated tumours. All patients provided written informed consent under a protocol approved by our Institutional Review Board. The average age of the patients was 64.5 (range: 39–90) years, 106 patients were men and 22 were women (ratio: 83:17). The primary locations of the tumours were the oral cavity (n = 44), hypopharynx (n = 32), larynx (n = 23), oropharynx (n = 23) and nasal cavity and paranasal sinus (n = 6).

RNA extraction and quantitative reverse-transcription polymerase chain reaction for determination of VEGFR mRNA levels

Total RNA was isolated using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and complementary DNA (cDNA) was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). The messenger RNA (mRNA) levels of VEGFR1, VEGFR2, VEGFR3 and glyceraldehyde 3-phosphate dehydrogenase were measured via quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) using SYBR Premix Ex Taq (Takara, Tokyo, Japan), the Takara Thermal Cycler Dice Real Time System TP8000 (Takara) and the primer sets presented in Supplemental Table 1. The results were analysed using the ^ΔΔCt method.

DNA isolation and quantitative methylation-specific PCR for determination of VEGFR methylation

Bisulphite modification of genomic DNA was carried out as described previously.²⁷ Methylation of the VEGFR and ACTB promoters was assessed via quantitative methylation-specific PCR (Q-MSP-PCR) using a SYBR Premix Dimer Eraser Kit (Takara), the Takara Thermal Cycler Dice Real Time System TP8000 and the primer sets shown in Supplemental Table 1. A standard curve was generated via serial dilutions of EpiScope Methylated HeLa genomic DNA (Takara). The normalized methylation value (NMV) was defined as follows: NMV = (VEGFR-S/VEGFR-FM)/(ACTB-S/ACTB-FM), where VEGFR-S and VEGFR-FM represent VEGFR methylation levels in the tumour sample and the universal methylated DNA standard, respectively, and ACTB-S and ACTB-FM correspond to the amount of ACTB (which encodes β-actin) in the tumour sample and the HeLa genomic DNA sample, respectively. The methylation status of the CpG islands in the promoter region of VEGFR1, VEGFR2 and VEGFR3 was determined in 128 primary HNSCC samples, 36 of which had matched noncancerous mucosal samples.

Analysis of publicly available data from The Cancer Genome Atlas

Aberrant DNA methylation data from the The Cancer Genome Atlas (TCGA) (available in November 2016) was collected from MethHC, a database of DNA methylation and gene expression in human cancer (http://methhc.mbc.nctu.edu.tw/php/index.php).²⁸ The DNA methylation data were collected using the Infinium HumanMethylation450 platform (Illumina, Inc., San Diego, CA, USA) and are presented as β values.

Data analysis and statistics

The Q-MSP results and patient characteristics (age of onset, gender, alcohol consumption, smoking status, tumour size, tumour stage, clinical stage, lymph node status and recurrence) were compared using Fisher’s exact probability test. Differences in VEGFR mRNA and VEGFR methylation levels between tumour tissue and adjacent normal tissue were evaluated using the paired t test. The methylation index (MI) was used to determine the overall methylation rate.²⁹ It was defined as the ratio of the number of methylated genes to the number of genes tested (in our case, 3).³⁰

The disease-free interval was measured from the date of treatment to the date at which locoregional recurrence or distant metastasis was diagnosed. Disease-free survival (DFS) probabilities were estimated using the Kaplan–Meier method, and the log-rank test was applied to assess the significance of differences between actuarial survival curves. Multivariate logistic regression analysis was used to determine the predictive values for recurrence and other variables according to methylation status. A difference was significant when the probability was less than 0.05. Statistical analysis was performed using Stat-Mate IV software (ATMS Co., Ltd, Tokyo, Japan).

Results

Initial screening: VEGFR expression and promoter methylation in HNSCC cell lines

The relative amounts of VEGFR1, VEGFR2 and VEGFR3 mRNA determined via Q-RT-PCR were significantly lower in cancer cell lines (UM-SCC-6, UM-SCC-47 and FaDu) than in normal tonsil samples (p = 0.057, p = 0.011 and p = 0.232, respectively) (Figure 1(a), (c) and (e)). In the Q-MSP analysis, the NMVs for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the cancer cells than in the normal tonsil samples (p = 0.068, p = 0.172 and p = 0.059, respectively) (Figure 1(b), (d) and (f)).

Figure 1.

VEGFR mRNA expression and VEGFR promoter methylation in head and neck squamous cell carcinoma cell lines. The relative amounts of (a) VEGFR1, (c) VEGFR2 and (e) VEGFR3 mRNA were lower in the cancer cell lines than in normal tonsil samples (p = 0.057, 0.011 and 0.232, respectively). The mean normalized methylation values (NMVs) for the (b) VEGFR1, (d) VEGFR2 and (f) VEGFR3 promoters tended to higher in the cancer cell lines than in normal tonsil samples (p = 0.068, 0.072 and 0.590, respectively). SCC: squamous cell carcinoma; NOS: normal tonsil sample.

Methylation patterns in HNSCCs and adjacent normal mucosal tissue

The NMVs of the VEGFR promoters were determined in 36 paired primary HNSCC samples and normal mucosal tissue samples. The methylation levels of the VEGFR1, VEGFR2 and VEGFR3 promoters were significantly higher in HNSCC tissue than in normal tissue (p = 0.048, p = 0.039 and p = 0.041, respectively) (Figure 2(a)–(c)). There was a trend towards decreased expression of VEGFR1, VEGFR2 and VEGF3 mRNA in HNSCC tissue compared with normal mucosal tissue (p = 0.045, p = 0.062 and p = 0.053, respectively) (Supplemental Figure 1).

Figure 2.

VEGFR methylation status in 36 matched pairs of head and neck tumour and normal tissue samples. The normalized methylation values (NMVs) for the (a) VEGFR1, (b) VEGFR2 and (c) VEGFR3 promoters were significantly higher in head and neck tumour tissue (T) than in paired adjacent normal mucosal tissue (N) (p = 0.048, 0.039 and 0.041, respectively).

Methylation status of the VEGFR promoters and MIs according to epidemiologic and clinical characteristics

A representative methylation analysis of the VEGFR genes in all 128 HNSCC samples is shown in Figure 3(a). The distribution of VEGFR1, VEGFR2 and VEGFR3 promoter methylation is shown in Figure 3(b). Methylation was observed in the promoters of all three VEGFR genes (MMM), two of three VEGFR genes (UMM, MMU, MUM), only one VEGFR gene (UUM, UMU, MUU) and none of the VEGFR genes (UUU) in 18%, 23%, 29% and 30% of the tumours, respectively (Figure 3(b)). We also compared the MIs according to epidemiologic and clinical characteristics. The MI was significantly higher in tumours in recurrence-positive (1.51 ± 1.05) compared with recurrence-negative (1.09 ± 1.09) cases (p = 0.029). There were no significant differences in MI in terms of age of onset, gender, alcohol consumption, smoking status, tumour stage, lymph node status or clinical stage (Figure 3(c)).

Figure 3.

Methylation status of the VEGFR promoters and methylation indexes (MIs) according to selected clinical parameters in 128 head and neck cancer squamous cell carcinoma samples. (a) A filled box indicates the presence of methylation, and an open box indicates the absence of methylation. (b) Distribution of VEGFR1, VEGFR2 and VEGFR3 promoter methylation. Methylation was observed in the promoters of all three VEGFR genes (MMM), two of three VEGFR genes (UMM, MMU, MUM), only one VEGFR gene ((UUM, UMU, MUU) and none of the VEGFR genes (UUU) in 18%, 23%, 29% and 30% of the tumours, respectively. (c) The mean MIs for the different groups were compared using Student’s t test. *p < 0.05. M: methylated; U: unmethylated.

Clinicopathologic characteristics of the 128 primary HNSCC samples

A DNA sample was classified as positive when the NMV exceeded 0.035, 0.078 and 0.018 for VEGFR1, VEGFR2 and VEGFR3, respectively. These cut-off values represented the mean NMV of the 36 normal mucosal tissue samples. The number of samples positive for VEGFR1, VEGFR2 and VEGFR3 promoter methylation was 66/128 (52%), 53/128 (41%) and 47/128 (37%), respectively (Figure 3(a), Table 1). Methylation of the VEGFR1 promoter (p = 0.003) and VEGFR3 promoter (p = 0.043) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046) (Table 1). There was no significant association between VEGFR1, VEGFR2 and VEGFR3 promoter methylation and HNSCC subtype (Supplemental Table 2). The subtypes in our study were oral (n = 44), hypopharynx (n = 32), laryngeal (n = 23), oropharyngeal (n = 23) and nasal cavity (n = 6).

Table 1.

VEGFR1, VEGFR2 and VEGFR3 gene methylation status in head and neck squamous cell carcinoma primary samples.

Patient and tumour characteristics	Methylation status
	VEGFR1			VEGFR2			VEGFR3
	Present (66)	Absent (62)	p value^a	Present (53)	Absent (75)	p value^a	Present (47)	Absent (81)	p value^a
Age
70 and older	19	19		18	20		12	26
Under 70	47	43	0.848	35	55	1	35	55	0.548
Gender
Male	55	51		40	66		39	67
Female	11	11	1	13	9	0.095	8	14	1
Alcohol exposure
Ever	41	45		37	49		31	55
Never	25	17	0.259	16	26	0.703	16	26	1
Smoking status
Smoker	46	47		38	55		33	60
Nonsmoker	20	15	0.552	15	20	1	14	21	1
Tumour size
T1–2	34	27		28	33		21	40
T3–4	32	35	0.382	25	42	0.371	26	41	0.714
Lymph node status
N0	27	28		17	38		19	36
N+	39	34	0.721	36	37	0.046*	28	45	0.713
Stage
I, II, III	29	26		23	32		19	36
IV	37	36	0.859	30	43	1	28	45	0.713
Recurrence events
Positive	41	22		25	38		29	34
Negative	25	40	0.003*	28	37	0.723	18	47	0.043*

Fisher’s exact probability test.

p < 0.05.

Prognostic value of VEGFR promoter methylation

The Kaplan–Meier survival curves for the 128 HNSCC patients according to the methylation status of the VEGFR1, VEGFR2 and VEGFR3 promoters are shown in Figure 4. Patients with methylated (compared with unmethylated) VEGFR1 and VEGFR3 promoters had a shorter DFS time (log-rank test; p = 0.034 and p = 0.026, respectively) (Figure 4(a) and (c)). A shorter DFS time was also observed when methylation of any the VEGFR promoters was compared with no methylation of all three VEGFR promoters (log-rank test, p = 0.017) (Figure 4(d)). Simultaneous VEGFR1 and VEGFR3 methylation correlated with worse survival in patients who underwent curative surgery (log-rank test, p = 0.009) (Figure 4(e)). Among the 55 patients without lymph node metastasis, those with a methylated VEGFR3 promoter had a shorter DFS time than did those with an unmethylated VEGFR3 promoter (log-rank test, p = 0.044) (Figure 4(f)).

Figure 4.

Kaplan–Meier survival curves according to VEGFR promoter methylation status in patients with head and neck squamous cell carcinoma. Disease-free survival according to (a) VEGFR1 methylation status, (b) VEGFR2 methylation status, (c) VEGFR3 methylation status, (d) VEGFR1, VEGFR2 and VEGFR3 methylation status, (e) VEGFR1 and VEGFR3 methylation status, (f) and VEGFR3 methylation status in patients without lymph node metastasis.

To assess the relationship between VEGFR promoter methylation and HNSCC recurrence, we performed a multivariate logistic regression analysis adjusted for age (≥70 vs <70 years), gender, smoking status, alcohol consumption and stage (I–III vs IV). The odds ratios for recurrence were 3.19 (95% confidence interval (CI): 1.51–6.75; p = 0.002), 2.24 (95% CI: 1.06–4.76; p = 0.035) and 2.56 (95% CI: 1.09–6.05; p = 0.032), for methylation of the VEGFR1, VEGFR3 and paired VEGFR1 and VEGFR3 promoters, respectively (Figure 5). The odds ratios for recurrence were also determined in a multivariate logistic regression analysis that also included HPV infection status as an adjustment factor (Supplemental Figure 2).

Figure 5.

Multivariate logistic regression analysis for recurrence. The analysis was adjusted for age (≥70 vs <70 years), gender, smoking status, alcohol consumption and tumour stage (I–III vs IV). The results show the estimated odds of recurrence associated with VEGFR1, VEGFR2 and VEGFR3 promoter methylation. *p < 0.05. CI: confidence interval.

Analysis of publicly available data from the TCGA

Aberrant promoter methylation of VEGFR1, VEGFR2 and VEGFR3 was detected in 516 HNSCC sample data compared with 50 normal samples (Supplemental Figure 3). The average β values for VEGFR1, VEGFR2 and VEGFR3 methylation were significantly higher in the HNSCC samples than in the normal samples (tumour values: 0.234, 0.226 and 0.245, respectively; normal values: 1.03, 0.084 and 0.117, respectively; all p < 0.05).

Discussion

The VEGFRs are members of the receptor tyrosine kinase (RTK) superfamily.⁷ They contain an extracellular ligand-binding domain, which consists of seven immunoglobulin-like folds, a single transmembrane region and a split tyrosine kinase domain.⁷ The VEGFRs activate the mitogen-activated protein kinase (MAPK) and AKT signalling pathways in the cytoplasm to promote cell proliferation and migration.^8,19,31

The promoters of all VEGFR genes contain CpG islands, which are associated with epigenetic silencing of gene expression in many cancer cell lines.^32,33 The efficacy of anti-VEGF/VEGFR drugs depends on the knowledge of epigenomic VEGFR modifications; for example, a previous study of VEGFR1 showed synergistic inhibition of renal cancer cell proliferation by a demethylating agent and an RTK inhibitor (sunitinib or axitinib).³⁴ The VEGFR2 promoter contains two GC-rich sites that are critical for its transcriptional activity. Methyl-CpG-binding protein 2 directly binds these sites when they are methylated and consequently represses VEGFR2 transcription.³⁵ In the VEGFR3 promoter, proximal GC-rich elements interact with Sp1 and Sp3 to activate transcription.^32,36 Methylation array analysis suggests that VEGFR2, VEGFR3 and TFPI2 (which encodes tissue factor pathway inhibitor 2) are useful biomarkers for early detection of oral SCC.²⁶

Negative signals generated by VEGFR1 and positive signals generated by VEGFR2 result in balanced blood vessel formation during early embryogenesis.³⁷ Both of these receptors also contribute to pathological angiogenesis and tumour growth and metastasis.⁸ Cancer stem cells derived from HNSCCs express VEGFR1 and are extremely vascularized,³⁸ and hypomethylation-related overexpression of VEGFR1 in the alveolar macrophages of smokers is thought to play a role in lung cancer initiation.³⁹ Increases in VEGFR1 abundance in intraepithelial neoplastic lesions and differentiated adenocarcinomas and decreases in VEGFR1 abundance in poorly differentiated carcinomas suggest activation of a molecular switch during prostate tumour progression.^40,41 VEGFR1 levels are higher in well-differentiated tumours and lower in poorly differentiated tumours and are associated with a lymph node–negative status in HNSCC.⁴²

Epigenetic alteration of the VEGFR genes is closely related to prognosis and response to chemotherapy. Using TCGA data derived from the MethHC database, we found that the VEGFR promoter was hypermethylated in samples from six tumour types including HNSCC compared to normal samples. Patients with solid tumours often systemically receive anti-VEGF drugs such as bevacizumab.⁴³ Anti-VEGF/VEGFR agents have been widely used for treatment of human cancers including colorectal, ovarian, breast, lung, glioblastoma, renal cell and cervical cancers.¹¹ A phase II trial evaluating the RTK sorafenib as a single agent in patients with recurrent or metastatic nasopharyngeal squamous cell carcinoma showed major clinical responses.⁴⁴ RTKs may be useful for management of HNSCC patients, particularly if combined with anti-VEGF/VEGFR drugs.

In this study, we systematically determined the methylation status of the promoters of three VEGFR genes and its relationship with clinical characteristics in 128 HNSCC samples. Methylation of all three VEGFR genes was detected in these samples. Methylation of the VEGFR1 and VEGFR3 promoters correlated with poor prognosis, whereas methylation of the VEGFR2 promoter correlated with lymph node metastasis.

In summary, methylation the VEGFR genes can predict lymph node metastasis and poor prognosis in HNSCC patients. Furthermore, determination of the levels of the VEGFR promoters will help select patients for targeted therapies. Although our findings support the use of methylation markers in clinical practice, they require further testing in prospective studies with larger HNSCC cohorts.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

All procedures involving surgical collection of tissue were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by a Grant-in-Aid for Scientific Research (nos 26462599, 26462600, 16K11228 and 16K20239) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Informed consent

Informed consent was obtained from the individuals from whom the tissue samples were derived.

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