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Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients.

Chou JL, Huang RL, Shay J, Chen LY, Lin SJ, Yan PS, Chao WT, Lai YH, Lai YL, Chao TK, Lee CI, Tai CK, Wu SF, Nephew KP, Huang TH, Lai HC, Chan MW - Clin Epigenetics (2015)

Bottom Line: The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing.Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019).Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Chung Cheng University, 168 University Road, Min-Hsiung, Chia-Yi 621, Taiwan ; Institute of Molecular Biology, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan ; Division of Gastroenterology, Chang Gung Memorial Hospital, Chia-Yi, Taiwan.

ABSTRACT

Background: The dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.

Results: We first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).

Conclusions: ABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

No MeSH data available.


Related in: MedlinePlus

Effects ofABCA1knockdown on cell growth. Real-time RT-PCR expression of ABCA1 in (A) MCP3 and (B) HeyC2 cells infected by lentivirus against shGFP (control) or shABCA1. Each bar represents mean ± SD. The growth of ABCA1 knockdown (C) MCP3 and (D) HeyC2 cells was examined by soft agar assay. Quantitative analysis of the soft agar assay is also shown. ***P < 0.001; *P < 0.05.
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Fig2: Effects ofABCA1knockdown on cell growth. Real-time RT-PCR expression of ABCA1 in (A) MCP3 and (B) HeyC2 cells infected by lentivirus against shGFP (control) or shABCA1. Each bar represents mean ± SD. The growth of ABCA1 knockdown (C) MCP3 and (D) HeyC2 cells was examined by soft agar assay. Quantitative analysis of the soft agar assay is also shown. ***P < 0.001; *P < 0.05.

Mentions: As ABCA1 is responsible for cholesterol efflux, we first measured the effect of ABCA1 depletion on the cholesterol level of ovarian cancer cells. Lentiviral knockdown of ABCA1 was performed in MCP3 (Figure 2A) and HeyC2 (Figure 2B) ovarian cancer cells. The result showed that more than 50% repression of ABCA1 was observed in these cells. As expected, depletion of ABCA1 resulted in a significant accumulation of cholesterol in MCP3 (Figure 3A, P < 0.05) and HeyC2 (Figure 3B, P < 0.05) cells.Figure 2


Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients.

Chou JL, Huang RL, Shay J, Chen LY, Lin SJ, Yan PS, Chao WT, Lai YH, Lai YL, Chao TK, Lee CI, Tai CK, Wu SF, Nephew KP, Huang TH, Lai HC, Chan MW - Clin Epigenetics (2015)

Effects ofABCA1knockdown on cell growth. Real-time RT-PCR expression of ABCA1 in (A) MCP3 and (B) HeyC2 cells infected by lentivirus against shGFP (control) or shABCA1. Each bar represents mean ± SD. The growth of ABCA1 knockdown (C) MCP3 and (D) HeyC2 cells was examined by soft agar assay. Quantitative analysis of the soft agar assay is also shown. ***P < 0.001; *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307187&req=5

Fig2: Effects ofABCA1knockdown on cell growth. Real-time RT-PCR expression of ABCA1 in (A) MCP3 and (B) HeyC2 cells infected by lentivirus against shGFP (control) or shABCA1. Each bar represents mean ± SD. The growth of ABCA1 knockdown (C) MCP3 and (D) HeyC2 cells was examined by soft agar assay. Quantitative analysis of the soft agar assay is also shown. ***P < 0.001; *P < 0.05.
Mentions: As ABCA1 is responsible for cholesterol efflux, we first measured the effect of ABCA1 depletion on the cholesterol level of ovarian cancer cells. Lentiviral knockdown of ABCA1 was performed in MCP3 (Figure 2A) and HeyC2 (Figure 2B) ovarian cancer cells. The result showed that more than 50% repression of ABCA1 was observed in these cells. As expected, depletion of ABCA1 resulted in a significant accumulation of cholesterol in MCP3 (Figure 3A, P < 0.05) and HeyC2 (Figure 3B, P < 0.05) cells.Figure 2

Bottom Line: The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing.Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019).Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Chung Cheng University, 168 University Road, Min-Hsiung, Chia-Yi 621, Taiwan ; Institute of Molecular Biology, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan ; Division of Gastroenterology, Chang Gung Memorial Hospital, Chia-Yi, Taiwan.

ABSTRACT

Background: The dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.

Results: We first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).

Conclusions: ABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

No MeSH data available.


Related in: MedlinePlus