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. 2018 Dec;7(12):1333-1342.
doi: 10.1530/EC-18-0380.

HOPX homeobox methylation in differentiated thyroid cancer and its clinical relevance

Erika Urbano Lima  1   2 , Ileana G S Rubio  1   2 , Joaquim Custodio Da Silva  3   4 , Ana Luiza Galrão  1 , Danielle Pêssoa  3   4 , Taise Cerqueira Oliveira  3 , Fabiane Carrijo  3   4 , Igor Silva Campos  5 , Luciano Fonseca Espinheira  5   6 , Luiz Jose Sampaio  7 , Claudio Rogerio Lima  8 , Janete Maria Cerutti  2   9 , Helton Estrela Ramos  3   4
Affiliations

HOPX homeobox methylation in differentiated thyroid cancer and its clinical relevance

Erika Urbano Lima et al. Endocr Connect. 2018 Dec.

Abstract

Background: The inactivation of the tumor-suppressor homeodomain-only protein X (HOPX) usually involves promoter methylation in several cancer types. This study aimed to investigate the HOPX-β mRNA expression and promoter methylation and their clinical relevance in differentiated thyroid cancer (DTC).

Patients and methods: Clinicopathological data and paraffin-embedded thyroid tumor tissues from 21 patients with DTC and 6 with benign tumors (T) and their non-tumor parenchyma (NT) were investigated. Tumor cell lines (FTC238, FTC236 and WRO) were treated with demethylating agent. HOPX-β mRNA expression was assessed by qRT-PCR and methylation status by Q-MSP. Thyroid cancer data from Cancer Genome Atlas (TCGA) was also collected.

Results: HOPX-β mRNA re-expression in two cell lines treated with demethylating agent was observed concomitantly with reduced promoter methylation. Reduced mRNA expression in T group compared to their NT was observed, and reduced protein expression in T compared to NT was observed in three cases. Low mRNA expression with high methylation status was detected in 6/14 DTC samples. High methylation status was associated with older age at diagnosis, recurrent or progressive disease and with the presence of new neoplasm event post initial therapy while hyper-methylation correlated with worse overall survival, worse disease-free status and older age.

Conclusion: A moderate coupling of downregulation of HOPX-β mRNA expression in DTC followed by high HOPX-β promoter methylation was observed however; high HOPX promoter methylation status was associated with the worse prognosis of DTC patients.

Keywords: HOPX; differentiated thyroid cancer; epigenetic; methylation.

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Figures

Figure 1
Figure 1
HOPX-β mRNA (A) and protein expression (C), and PMR (percent of methylated reference) values of HOPXβ promoter methylation (B) before (DMSO) and after (5AZA) treatment with the demethylating agent 5-AzadC in cell cultures of FTC236, FTC 238 and WRO. (*P = 0.03 between control and treated cells.) AU, arbitrary units.
Figure 2
Figure 2
Expression level of HOPX-β mRNA in different primary thyroid tumors (A) malignant group, n = 21; (B) benign group, n = 6; and their matched non-tumor tissues determined by qRT-PCR. Reduced expression in tumor compared with non-tumor samples was observed in malignant groups (*P = 0.03). AU, arbitrary units.
Figure 3
Figure 3
PMR (percent of methylated reference) values of HOPX-β promoter methylation in differentiated thyroid cancer (tumor) (n = 14) and their matched non-tumor tissues determined by Q-MSP (quantitative methylated PCR). A tendency of high degree of methylation was observed in the malignant tumor samples compared to the matched surrounding non-tumor samples (P = 0.055). AU, arbitrary units.
Figure 4
Figure 4
HOPX-β mRNA (top panel) and PMR (percent of methylated reference) values of HOPXβ promoter methylation (bottom panel) in 14/19 pairs of paraffin-embedded DTC samples. AU, arbitrary units; P, patient. Black bar: non-tumor, gray bar: tumor.
Figure 5
Figure 5
Western blot for HOPX protein expression in three pairs tissue samples (T/NT), 22, 23 and 24 with primary HOPX antibody (1:1000; Cell Signaling) showed reduced expression in the T samples compared with NT samples. Tubulin expression was used as control (1:10,000; Sigma).
Figure 6
Figure 6
HOPX-β methylation values in tumor (T) and non-tumor (NT) groups in all thyroid cancer TCGA samples (A) and in paired samples (B), *P < 0.0005.
Figure 7
Figure 7
Kaplan–Meier survival analysis using hyper-methylation threshold value of 1.89 showed significant association of HOPX promoter methylation with diagnosis age, disease free status and overall survival (P = 0.006, P = 0.008, P = 0.03, respectively).
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