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Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.

Korah R, Healy JM, Kunstman JW, Fonseca AL, Ameri AH, Prasad ML, Carling T - Mol. Cancer (2013)

Bottom Line: Using adrenocortical tumor and normal tissue specimens, we show a significant reduction in expression of RASSF1A mRNA and protein in ACC.Conversely, the RASSF1A promoter methylation profile in benign adrenocortical adenomas (ACAs) was found to be very similar to that found in normal adrenal cortex.On the other hand, expression of RASSF1A/A133S, a loss-of-function mutant form of RASSF1A, failed to elicit similar malignancy-suppressing responses in ACC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT

Background: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with high mutational heterogeneity and a generally poor clinical outcome. Despite implicated roles of deregulated TP53, IGF-2 and Wnt signaling pathways, a clear genetic association or unique mutational link to the disease is still missing. Recent studies suggest a crucial role for epigenetic modifications in the genesis and/or progression of ACC. This study specifically evaluates the potential role of epigenetic silencing of RASSF1A, the most commonly silenced tumor suppressor gene, in adrenocortical malignancy.

Results: Using adrenocortical tumor and normal tissue specimens, we show a significant reduction in expression of RASSF1A mRNA and protein in ACC. Methylation-sensitive and -dependent restriction enzyme based PCR assays revealed significant DNA hypermethylation of the RASSF1A promoter, suggesting an epigenetic mechanism for RASSF1A silencing in ACC. Conversely, the RASSF1A promoter methylation profile in benign adrenocortical adenomas (ACAs) was found to be very similar to that found in normal adrenal cortex. Enforced expression of ectopic RASSF1A in the SW-13 ACC cell line reduced the overall malignant behavior of the cells, which included impairment of invasion through the basement membrane, cell motility, and solitary cell survival and growth. On the other hand, expression of RASSF1A/A133S, a loss-of-function mutant form of RASSF1A, failed to elicit similar malignancy-suppressing responses in ACC cells. Moreover, association of RASSF1A with the cytoskeleton in RASSF1A-expressing ACC cells and normal adrenal cortex suggests a role for RASSF1A in modulating microtubule dynamics in the adrenal cortex, and thereby potentially blocking malignant progression.

Conclusions: Downregulation of RASSF1A via promoter hypermethylation may play a role in the malignant progression of adrenocortical carcinoma possibly by abrogating differentiation-promoting RASSF1A- microtubule interactions.

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RASSF1A expression and promoter methylation in ACC cell lines. (A) Indirect immunofluorescence detection of RASSF1A protein (FITC – green) expression in SW-13, NCI-H295R and ACT-1 (a thyroid cancer cell line used as a positive control for RASSF1A expression) cells. Cell nuclei fluoresces blue due to DAPI fluorescence. (B) RASSF1A promoter methylation pattern in exponentially growing cultures of NCI-H295R and SW-13 cells as determined by Epitect methyl II PCR assay. Averages of percentage Hypermethylated (FHM) intermediate methylated (FIM), and unmethylated (FUM) CpGs are shown. (C &D) SW-13 cells were grown in the presence of varying (0, 0.1, 1, 5 and 10 μM) concentrations of 5-aza-2’-deoxycitidine for 48 hours and (C) Epitect methyl II assay was performed on genomic DNA to determine RASSF1A promoter methylation, and (D) RASSF1A mRNA expression was assayed by real-time qPCR. Average mRNA expression values of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP) were used for normalization.
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Figure 2: RASSF1A expression and promoter methylation in ACC cell lines. (A) Indirect immunofluorescence detection of RASSF1A protein (FITC – green) expression in SW-13, NCI-H295R and ACT-1 (a thyroid cancer cell line used as a positive control for RASSF1A expression) cells. Cell nuclei fluoresces blue due to DAPI fluorescence. (B) RASSF1A promoter methylation pattern in exponentially growing cultures of NCI-H295R and SW-13 cells as determined by Epitect methyl II PCR assay. Averages of percentage Hypermethylated (FHM) intermediate methylated (FIM), and unmethylated (FUM) CpGs are shown. (C &D) SW-13 cells were grown in the presence of varying (0, 0.1, 1, 5 and 10 μM) concentrations of 5-aza-2’-deoxycitidine for 48 hours and (C) Epitect methyl II assay was performed on genomic DNA to determine RASSF1A promoter methylation, and (D) RASSF1A mRNA expression was assayed by real-time qPCR. Average mRNA expression values of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP) were used for normalization.

Mentions: To investigate the functional significance of promoter hypermethylation and consequent RASSF1A silencing in adrenocortical carcinogenesis, we sought to utilize a cell culture model. To identify a suitable model, we analyzed the RASSF1A expression pattern in two widely used ACC cell lines NCI-H295R and SW-13, by indirect immunofluorescence (Figure 2A). As shown in Figure 2A, both ACC cell lines revealed undetectable levels of RASSF1A, when compared with the expression in a thyroid cancer cell line ACT-1. Next, we examined the methylation pattern of NCI-H295R and SW-13 cells which showed very high levels of methylation in both cell types (Figure 2B). However, the methylation pattern appeared to be different between the two ACC cell lines. While NCI-H295R cells showed no hypermethylation, similar to ACA and normal adrenal tissue methylation, SW-13 cells showed more than 99% hypermethylation in the RASSF1 promoter (Figure 2B), similar to the hypermethylation levels observed in some ACC samples (note Figure 1B). Therefore, we chose SW-13 cells for further functional studies. To confirm RASSF1A promoter hypermethylation as the cause of RASSF1A downregulation in SW-13 cells, we treated the cells with a widely used de-methylating agent 5-aza-2’-deoxycytidine[35]. After 48 hours of treatment with 5-aza-2’-deoxycytidine, RASSF1A promoter analysis showed a dose-dependent reversal of hypermethylation (Figure 2C) and a consequent dose-dependent increase in the expression levels of RASSF1A mRNA (Figure 2D).


Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.

Korah R, Healy JM, Kunstman JW, Fonseca AL, Ameri AH, Prasad ML, Carling T - Mol. Cancer (2013)

RASSF1A expression and promoter methylation in ACC cell lines. (A) Indirect immunofluorescence detection of RASSF1A protein (FITC – green) expression in SW-13, NCI-H295R and ACT-1 (a thyroid cancer cell line used as a positive control for RASSF1A expression) cells. Cell nuclei fluoresces blue due to DAPI fluorescence. (B) RASSF1A promoter methylation pattern in exponentially growing cultures of NCI-H295R and SW-13 cells as determined by Epitect methyl II PCR assay. Averages of percentage Hypermethylated (FHM) intermediate methylated (FIM), and unmethylated (FUM) CpGs are shown. (C &D) SW-13 cells were grown in the presence of varying (0, 0.1, 1, 5 and 10 μM) concentrations of 5-aza-2’-deoxycitidine for 48 hours and (C) Epitect methyl II assay was performed on genomic DNA to determine RASSF1A promoter methylation, and (D) RASSF1A mRNA expression was assayed by real-time qPCR. Average mRNA expression values of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP) were used for normalization.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: RASSF1A expression and promoter methylation in ACC cell lines. (A) Indirect immunofluorescence detection of RASSF1A protein (FITC – green) expression in SW-13, NCI-H295R and ACT-1 (a thyroid cancer cell line used as a positive control for RASSF1A expression) cells. Cell nuclei fluoresces blue due to DAPI fluorescence. (B) RASSF1A promoter methylation pattern in exponentially growing cultures of NCI-H295R and SW-13 cells as determined by Epitect methyl II PCR assay. Averages of percentage Hypermethylated (FHM) intermediate methylated (FIM), and unmethylated (FUM) CpGs are shown. (C &D) SW-13 cells were grown in the presence of varying (0, 0.1, 1, 5 and 10 μM) concentrations of 5-aza-2’-deoxycitidine for 48 hours and (C) Epitect methyl II assay was performed on genomic DNA to determine RASSF1A promoter methylation, and (D) RASSF1A mRNA expression was assayed by real-time qPCR. Average mRNA expression values of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP) were used for normalization.
Mentions: To investigate the functional significance of promoter hypermethylation and consequent RASSF1A silencing in adrenocortical carcinogenesis, we sought to utilize a cell culture model. To identify a suitable model, we analyzed the RASSF1A expression pattern in two widely used ACC cell lines NCI-H295R and SW-13, by indirect immunofluorescence (Figure 2A). As shown in Figure 2A, both ACC cell lines revealed undetectable levels of RASSF1A, when compared with the expression in a thyroid cancer cell line ACT-1. Next, we examined the methylation pattern of NCI-H295R and SW-13 cells which showed very high levels of methylation in both cell types (Figure 2B). However, the methylation pattern appeared to be different between the two ACC cell lines. While NCI-H295R cells showed no hypermethylation, similar to ACA and normal adrenal tissue methylation, SW-13 cells showed more than 99% hypermethylation in the RASSF1 promoter (Figure 2B), similar to the hypermethylation levels observed in some ACC samples (note Figure 1B). Therefore, we chose SW-13 cells for further functional studies. To confirm RASSF1A promoter hypermethylation as the cause of RASSF1A downregulation in SW-13 cells, we treated the cells with a widely used de-methylating agent 5-aza-2’-deoxycytidine[35]. After 48 hours of treatment with 5-aza-2’-deoxycytidine, RASSF1A promoter analysis showed a dose-dependent reversal of hypermethylation (Figure 2C) and a consequent dose-dependent increase in the expression levels of RASSF1A mRNA (Figure 2D).

Bottom Line: Using adrenocortical tumor and normal tissue specimens, we show a significant reduction in expression of RASSF1A mRNA and protein in ACC.Conversely, the RASSF1A promoter methylation profile in benign adrenocortical adenomas (ACAs) was found to be very similar to that found in normal adrenal cortex.On the other hand, expression of RASSF1A/A133S, a loss-of-function mutant form of RASSF1A, failed to elicit similar malignancy-suppressing responses in ACC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT

Background: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with high mutational heterogeneity and a generally poor clinical outcome. Despite implicated roles of deregulated TP53, IGF-2 and Wnt signaling pathways, a clear genetic association or unique mutational link to the disease is still missing. Recent studies suggest a crucial role for epigenetic modifications in the genesis and/or progression of ACC. This study specifically evaluates the potential role of epigenetic silencing of RASSF1A, the most commonly silenced tumor suppressor gene, in adrenocortical malignancy.

Results: Using adrenocortical tumor and normal tissue specimens, we show a significant reduction in expression of RASSF1A mRNA and protein in ACC. Methylation-sensitive and -dependent restriction enzyme based PCR assays revealed significant DNA hypermethylation of the RASSF1A promoter, suggesting an epigenetic mechanism for RASSF1A silencing in ACC. Conversely, the RASSF1A promoter methylation profile in benign adrenocortical adenomas (ACAs) was found to be very similar to that found in normal adrenal cortex. Enforced expression of ectopic RASSF1A in the SW-13 ACC cell line reduced the overall malignant behavior of the cells, which included impairment of invasion through the basement membrane, cell motility, and solitary cell survival and growth. On the other hand, expression of RASSF1A/A133S, a loss-of-function mutant form of RASSF1A, failed to elicit similar malignancy-suppressing responses in ACC cells. Moreover, association of RASSF1A with the cytoskeleton in RASSF1A-expressing ACC cells and normal adrenal cortex suggests a role for RASSF1A in modulating microtubule dynamics in the adrenal cortex, and thereby potentially blocking malignant progression.

Conclusions: Downregulation of RASSF1A via promoter hypermethylation may play a role in the malignant progression of adrenocortical carcinoma possibly by abrogating differentiation-promoting RASSF1A- microtubule interactions.

Show MeSH
Related in: MedlinePlus