Methylation of TMEM176A, a key ERK Signaling Regulator, is a Novel Synthetic Lethality Marker of ATM Inhibitors in Human Lung Cancer

Figures & data

Figure 1. Methylation regulation of TMEM176A in human lung cancer cells.

(A) The association of TMEM176A expression and methylation at each CpG site of promoter region. (B) Scatter plots show reduced TMEM176A expression is associated with methylation status of cg19336959 in 457 cases of lung adenocarcinoma and 372 cases of lung squamous cell carcinoma. (C) Semiquantitative reverse transciptase PCR and western blot show the levels of TMEM176A expression in lung cancer cells (H157, H358, H1299, H23, H1563, A549, H446, H460 and H727) both in RNA and protein levels. (D) Methylation status of TMEM176A in lung cancer cells. (E) BSSQ results in H1299, H23, H727 cells and normal lung. Filled circles: methylation; open circles: unmethylation.

***p < 0.001.

BSSQ: Sodium bisulfite sequence; (-): without DAC; (+): with DAC; AD: Lung adenocarcinoma; GAPDH: Internal control; H2O: Double-distilled water; IVD: Invitral methylated DNA; M: Methylation; MSP: Methylation-specific PCR; NL: Normal lymph cell DNA; SCC: Lung squamous cell carcinoma; TSS: Transcription start site; U: Unmethylation.

Figure 2. Methylation status and TMEM176A staining in primary lung cancer.

(A) Representative methylation-specific PCR results. (B) TMEM176A staining in lung cancer and adjacent tissue samples (top: 200×; bottom: 400×). (C) Box plots for TMEM176A expression, horizontal lines represent the median score; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; vertical bars represent expression levels. (D) Bar diagram: the levels of TMEM176A expression and methylation status.

*p < 0.05; ***p < 0.001.

IVD: In vitro methylated DNA; LC: Primary lung cancer; M: Methylation; N: Normal lung tissue samples; NL: Normal lymph cell DNA; U: Unmethylation.

Table 1. The association of TMEM176A methylation and clinical factors in non-small-cell lung cancer.

Clinical factor	Number	TMEM176A methylation status		p-value^†
		Unmethylated n = 57 (46.34%)	Methylated n = 66 (53.66%)
Age (years)				0.412
– <60	62	31	31
– ≥60	61	26	35
Gender				0.879
– Male	92	43	49
– Female	31	14	17
Smoking				0.529
– Yes	77	34	43
– No	46	23	23
Alcohol abuse				0.125
– Yes	41	15	26
– No	82	42	40
Tumor size (cm)				0.137
– >5	12	8	4
– ≤5	111	49	62
Differentiation				0.196
– Well	24	14	10
– Moderate	73	29	44
– Poor	26	14	12
TNM stage				0.468
– Stage I	41	17	24
– Stage II	41	23	18
– Stage III	35	15	20
– Stage IV	6	2	4
Lymph node metastasis				0.646
– Negative	62	30	32
– Positive	61	27	34

† p-values are obtained from chi-square test.

TNM: Tumor-node-metastasis.

Figure 3. The role of TMEM176A in lung cancer cell proliferation, apoptosis, invasion and migration.

(A) Growth curves represent cell viability. (B) Colony formation results. Bar diagram: average number of tumor clones. (C) Flow cytometry shows apoptosis results. (D) The effect of TMEM176A on caspase-3, cleaved caspase-3, Bcl2 and Bax expression in H23, H1299 and H727 cells. siRNA#1 and siRNA#2: siRNA for TMEM176A. (E) Cell phase distribution in TMEM176A unexpressed and expressed H23, H1299 and H727 cells. (F) The effect of TMEM176A on CDC2 and cyclin B1 expression in H23, H1299 and H727 cells. (G) The migration and invasion assays for TMEM176A expressed and unexpressed H23 and H1299 cells. (H) The effect of TMEM176A on MMP-2 and MMP-9 expression in H23 and H1299 cells.

*p < 0.05; **p < 0.01; ***p < 0.001.

β-actin: Internal control; Scrambled: Scrambled siRNA serve as negative control; siRNA#2: siRNA for TMEM176A; TMEM176A: TMEM176A expressing vector; Vector: Control vector.

Figure 4. TMEM176A methylation activated the ERK signaling and sensitized lung cancer cells to AZD0156.

(A) western blots results. β-actin: internal control. (-): no serum stimulation; (+): serum stimulation. (B) Growth curves represent cell viability. SiRNA#2: siRNA for TMEM176A. (C) western blot results. SCH772984: p-ERK1/2 inhibitor; (-): absence of SCH772984; (+): presence of SCH772984. (D) MTT assays show OD value for IC₅₀. ATMi: ATM inhibitor. (E) Epigenetic silencing TMEM176A sensitized lung cancer cells to UV and AZD0156. western blot shows the levels of TMEM176A, ATM, p-CHK2, γ-H2AX, RAD51 and β-actin.

***p < 0.001.

AZD0156: ATM inhibitor; (-): absence of AZD0156 or UV; (+): presence of AZD0156 or UV.

Figure 5. The effect of TMEM176A on MMP-2, MMP-9 and p-ERK1/2 in H1299 cell xenografts.

(A) TMEM176A unexpressed and re-expressed H1299 cell xenografts in mice. (B) Growth curves for TMEM176A unexpressed and re-expressed H1299 cell xenografts. (C) Tumor weights: TMEM176A unexpressed and re-expressed H1299 cell xenografts. (D) TMEM176A affects the levels of MMP-2, MMP-9 and p-ERK1/2 in H1299 cell xenografts.

***p < 0.001.

Figure 6. A working model of synthetic lethality for ATM inhibitors and epigenetic silencing TMEM176A in lung cancer cells.