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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 regulation in adrenal tumor. (A) Averages of percentage methylated (FM) and unmethylated (FUM) CpGs in CpG island A of RASSF1A promoters in Normal adrenal cortex (n = 6), ACA (n = 8), and ACC (n = 7) samples are shown. FM includes both Hypermethylated (FHM) and intermediate methylated (FIM) fractions. (B) Methylation profiles of individual fresh-frozen normal adrenal cortex (N1 – N6), 8 ACAs (A1 – A8) and 7 ACC samples (C1 – C7) as determined by Epitect methyl II PCR assay. (C) Expression of RASSF1A mRNA determined by real-time qPCR in 7 ACC samples (C1 – C7) compared to the average expression in 6 normal samples (N) normalized to a value of 1.0. RASSF1A expressions in individual samples were also normalized to the average mRNA expression of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP). C-Av represents the average expression of all ACC samples. Data shown is from one of triplicate experiments that yielded similar results (mean ± SD). Independent sample t-test used to derive the p value (p = <0.01). (D) RASSF1A protein expression in normal (a &b) and ACC (c &d) FFPE tissue specimens demonstrated by immunohistochemistry through DAB staining (brown indicates RASSF1A protein expression) followed by nuclear counterstaining by hematoxylin (blue).
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Figure 1: RASSF1A expression and regulation in adrenal tumor. (A) Averages of percentage methylated (FM) and unmethylated (FUM) CpGs in CpG island A of RASSF1A promoters in Normal adrenal cortex (n = 6), ACA (n = 8), and ACC (n = 7) samples are shown. FM includes both Hypermethylated (FHM) and intermediate methylated (FIM) fractions. (B) Methylation profiles of individual fresh-frozen normal adrenal cortex (N1 – N6), 8 ACAs (A1 – A8) and 7 ACC samples (C1 – C7) as determined by Epitect methyl II PCR assay. (C) Expression of RASSF1A mRNA determined by real-time qPCR in 7 ACC samples (C1 – C7) compared to the average expression in 6 normal samples (N) normalized to a value of 1.0. RASSF1A expressions in individual samples were also normalized to the average mRNA expression of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP). C-Av represents the average expression of all ACC samples. Data shown is from one of triplicate experiments that yielded similar results (mean ± SD). Independent sample t-test used to derive the p value (p = <0.01). (D) RASSF1A protein expression in normal (a &b) and ACC (c &d) FFPE tissue specimens demonstrated by immunohistochemistry through DAB staining (brown indicates RASSF1A protein expression) followed by nuclear counterstaining by hematoxylin (blue).

Mentions: RASSF1 CpG island A hypermethylation is the most common epigenetic mechanism observed in tumors with silenced RASSF1A function[31,32,46,47]. To test whether promoter hypermethylation and consequent RASSF1A silencing contributes to adrenocortical tumorigenesis, we first determined the methylation status of CpG island A of RASSF1 in fresh-frozen ACC (n = 7), ACA (adrenocortical adenoma; n = 8) and normal adrenal cortex (n = 6) tissue specimens. Rarity of the disease and scarcity of adequate amounts of the specimens for assays constrained us from recruiting a larger cohort. We used a methylation-sensitive and -dependent restriction digestion based qPCR strategy to evaluate the methylation status of the 737bp area that spans RASSF1 CpG island A. This technique enables qualitative characterization (i.e. – regions demonstrating hypo-/intermediate-/hyper- methylation) of DNA methylation. The overall methylation profiles of normal and benign ACA samples were found to be very similar (57% and 60% respectively) while malignant ACCs showed a distinct statistically significant increase (86%) in the methylated fraction (Figure 1A). Analysis of methylation patterns in individual samples showed very low levels of hypermethylation (which represents >60% digestion by methylation-dependent restriction enzyme) in all normal and ACA cases (Figure 1B). Hypermethylation in normal adrenal cortex samples ranged from 0.2 – 2.0 % with an average of 0.35% and ACA samples ranged from 0.03 – 1.7% with an average hypermethylation of 0.7%. Conversely, all the ACC samples tested showed hypermethylation in excess of the maximum level observed in normal and ACA tissues. About 60% (4/7) of ACC samples had very high (>20%) hypermethylation of the CpG island A of the RASSF1 promoter (Figure 1B).


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 regulation in adrenal tumor. (A) Averages of percentage methylated (FM) and unmethylated (FUM) CpGs in CpG island A of RASSF1A promoters in Normal adrenal cortex (n = 6), ACA (n = 8), and ACC (n = 7) samples are shown. FM includes both Hypermethylated (FHM) and intermediate methylated (FIM) fractions. (B) Methylation profiles of individual fresh-frozen normal adrenal cortex (N1 – N6), 8 ACAs (A1 – A8) and 7 ACC samples (C1 – C7) as determined by Epitect methyl II PCR assay. (C) Expression of RASSF1A mRNA determined by real-time qPCR in 7 ACC samples (C1 – C7) compared to the average expression in 6 normal samples (N) normalized to a value of 1.0. RASSF1A expressions in individual samples were also normalized to the average mRNA expression of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP). C-Av represents the average expression of all ACC samples. Data shown is from one of triplicate experiments that yielded similar results (mean ± SD). Independent sample t-test used to derive the p value (p = <0.01). (D) RASSF1A protein expression in normal (a &b) and ACC (c &d) FFPE tissue specimens demonstrated by immunohistochemistry through DAB staining (brown indicates RASSF1A protein expression) followed by nuclear counterstaining by hematoxylin (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750604&req=5

Figure 1: RASSF1A expression and regulation in adrenal tumor. (A) Averages of percentage methylated (FM) and unmethylated (FUM) CpGs in CpG island A of RASSF1A promoters in Normal adrenal cortex (n = 6), ACA (n = 8), and ACC (n = 7) samples are shown. FM includes both Hypermethylated (FHM) and intermediate methylated (FIM) fractions. (B) Methylation profiles of individual fresh-frozen normal adrenal cortex (N1 – N6), 8 ACAs (A1 – A8) and 7 ACC samples (C1 – C7) as determined by Epitect methyl II PCR assay. (C) Expression of RASSF1A mRNA determined by real-time qPCR in 7 ACC samples (C1 – C7) compared to the average expression in 6 normal samples (N) normalized to a value of 1.0. RASSF1A expressions in individual samples were also normalized to the average mRNA expression of house-keeping genes beta-actin (Actb) and TATA-binding protein (TBP). C-Av represents the average expression of all ACC samples. Data shown is from one of triplicate experiments that yielded similar results (mean ± SD). Independent sample t-test used to derive the p value (p = <0.01). (D) RASSF1A protein expression in normal (a &b) and ACC (c &d) FFPE tissue specimens demonstrated by immunohistochemistry through DAB staining (brown indicates RASSF1A protein expression) followed by nuclear counterstaining by hematoxylin (blue).
Mentions: RASSF1 CpG island A hypermethylation is the most common epigenetic mechanism observed in tumors with silenced RASSF1A function[31,32,46,47]. To test whether promoter hypermethylation and consequent RASSF1A silencing contributes to adrenocortical tumorigenesis, we first determined the methylation status of CpG island A of RASSF1 in fresh-frozen ACC (n = 7), ACA (adrenocortical adenoma; n = 8) and normal adrenal cortex (n = 6) tissue specimens. Rarity of the disease and scarcity of adequate amounts of the specimens for assays constrained us from recruiting a larger cohort. We used a methylation-sensitive and -dependent restriction digestion based qPCR strategy to evaluate the methylation status of the 737bp area that spans RASSF1 CpG island A. This technique enables qualitative characterization (i.e. – regions demonstrating hypo-/intermediate-/hyper- methylation) of DNA methylation. The overall methylation profiles of normal and benign ACA samples were found to be very similar (57% and 60% respectively) while malignant ACCs showed a distinct statistically significant increase (86%) in the methylated fraction (Figure 1A). Analysis of methylation patterns in individual samples showed very low levels of hypermethylation (which represents >60% digestion by methylation-dependent restriction enzyme) in all normal and ACA cases (Figure 1B). Hypermethylation in normal adrenal cortex samples ranged from 0.2 – 2.0 % with an average of 0.35% and ACA samples ranged from 0.03 – 1.7% with an average hypermethylation of 0.7%. Conversely, all the ACC samples tested showed hypermethylation in excess of the maximum level observed in normal and ACA tissues. About 60% (4/7) of ACC samples had very high (>20%) hypermethylation of the CpG island A of the RASSF1 promoter (Figure 1B).

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