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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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Constitutive expression of RASSF1A reduces the invasive, migratory and clonogenic potentials of SW-13 cells. (A), SW-13/A and SW-13/AM cells were allowed to invade through Matrigel from upper chambers containing serum-free medium to lower chambers containing 10% FBS medium. After 24 hours, and invaded cells were fixed, stained with crystal violet and tabulated. Data represent results from one of two independent experiments with similar results. (B) SW-13/V, SW-13/A and SW-13/AM cells were allowed to migrate through modified Boyden Chambers (8 μM pore size) for 4 hours and migrated cells to the lower side of the membrane were fixed, stained with crystal violet and tabulated. Data from a representative experiment of triplicate experiments with similar results are shown. (C) SW-13/V, SW-13/A and SW-13/AM cells were seeded in 6-well plates in low densities (5000 cells/well) and allowed to grow for 7 days in G-418 containing medium. Cells were washed with PBS, fixed in 3.7 % formaldehyde solution stained with crystal violet and colonies with 10 +/− 2 cells were counted and averaged from 6 wells. Data from a representative experiment of quadruplicate experiments with similar results are shown.
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Figure 5: Constitutive expression of RASSF1A reduces the invasive, migratory and clonogenic potentials of SW-13 cells. (A), SW-13/A and SW-13/AM cells were allowed to invade through Matrigel from upper chambers containing serum-free medium to lower chambers containing 10% FBS medium. After 24 hours, and invaded cells were fixed, stained with crystal violet and tabulated. Data represent results from one of two independent experiments with similar results. (B) SW-13/V, SW-13/A and SW-13/AM cells were allowed to migrate through modified Boyden Chambers (8 μM pore size) for 4 hours and migrated cells to the lower side of the membrane were fixed, stained with crystal violet and tabulated. Data from a representative experiment of triplicate experiments with similar results are shown. (C) SW-13/V, SW-13/A and SW-13/AM cells were seeded in 6-well plates in low densities (5000 cells/well) and allowed to grow for 7 days in G-418 containing medium. Cells were washed with PBS, fixed in 3.7 % formaldehyde solution stained with crystal violet and colonies with 10 +/− 2 cells were counted and averaged from 6 wells. Data from a representative experiment of quadruplicate experiments with similar results are shown.

Mentions: Advanced features of malignancy, such as invasion of or migration to adjacent tissues, degradation of the extra cellular matrix (ECM), and clonogenic survival and growth, are hallmarks of aggressive tumors such as ACC that portend a poor clinical outcome. We evaluated whether constitutive expression of RASSF1A have any effect on the malignant phenotype of SW-13 cells. Cell invasiveness was evaluated using an overnight Matrigel invasion assay, which showed a significant reduction in the invasive potential of SW-13 cells constitutively expressing RASSF1A (SW-13/A) compared to empty vector (SW-13/V) or RASSF1A mutant (SW-13/AM) (Figure 5A). To test whether the reduced invasive potential is through an impaired migratory response, cells were allowed to migrate through 8μm pore-carrying cell culture inserts following a nutrient gradient. After 4 hours, SW-13 cells expressing RASSF1A showed a 5-fold reduction in the number of cells migrated across the membrane (Figure 5B), suggesting a strong motility-inhibitory response from re-expressed RASSF1A. Similarly, cells constitutively expressing high levels of RASSF1A also showed a significantly impaired clonogenic survival/growth response when compared to cells not expressing RASSF1A or cells expressing high levels of RASSF1A/A133S mutant proteins (Figure 5C). In summary, RASSF1A expression resulted in an overall reduced malignant behavior of SW-13 ACC cells which was not observed in cells expressing the RASSF1A/A133S mutant protein.


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)

Constitutive expression of RASSF1A reduces the invasive, migratory and clonogenic potentials of SW-13 cells. (A), SW-13/A and SW-13/AM cells were allowed to invade through Matrigel from upper chambers containing serum-free medium to lower chambers containing 10% FBS medium. After 24 hours, and invaded cells were fixed, stained with crystal violet and tabulated. Data represent results from one of two independent experiments with similar results. (B) SW-13/V, SW-13/A and SW-13/AM cells were allowed to migrate through modified Boyden Chambers (8 μM pore size) for 4 hours and migrated cells to the lower side of the membrane were fixed, stained with crystal violet and tabulated. Data from a representative experiment of triplicate experiments with similar results are shown. (C) SW-13/V, SW-13/A and SW-13/AM cells were seeded in 6-well plates in low densities (5000 cells/well) and allowed to grow for 7 days in G-418 containing medium. Cells were washed with PBS, fixed in 3.7 % formaldehyde solution stained with crystal violet and colonies with 10 +/− 2 cells were counted and averaged from 6 wells. Data from a representative experiment of quadruplicate experiments with similar results are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Constitutive expression of RASSF1A reduces the invasive, migratory and clonogenic potentials of SW-13 cells. (A), SW-13/A and SW-13/AM cells were allowed to invade through Matrigel from upper chambers containing serum-free medium to lower chambers containing 10% FBS medium. After 24 hours, and invaded cells were fixed, stained with crystal violet and tabulated. Data represent results from one of two independent experiments with similar results. (B) SW-13/V, SW-13/A and SW-13/AM cells were allowed to migrate through modified Boyden Chambers (8 μM pore size) for 4 hours and migrated cells to the lower side of the membrane were fixed, stained with crystal violet and tabulated. Data from a representative experiment of triplicate experiments with similar results are shown. (C) SW-13/V, SW-13/A and SW-13/AM cells were seeded in 6-well plates in low densities (5000 cells/well) and allowed to grow for 7 days in G-418 containing medium. Cells were washed with PBS, fixed in 3.7 % formaldehyde solution stained with crystal violet and colonies with 10 +/− 2 cells were counted and averaged from 6 wells. Data from a representative experiment of quadruplicate experiments with similar results are shown.
Mentions: Advanced features of malignancy, such as invasion of or migration to adjacent tissues, degradation of the extra cellular matrix (ECM), and clonogenic survival and growth, are hallmarks of aggressive tumors such as ACC that portend a poor clinical outcome. We evaluated whether constitutive expression of RASSF1A have any effect on the malignant phenotype of SW-13 cells. Cell invasiveness was evaluated using an overnight Matrigel invasion assay, which showed a significant reduction in the invasive potential of SW-13 cells constitutively expressing RASSF1A (SW-13/A) compared to empty vector (SW-13/V) or RASSF1A mutant (SW-13/AM) (Figure 5A). To test whether the reduced invasive potential is through an impaired migratory response, cells were allowed to migrate through 8μm pore-carrying cell culture inserts following a nutrient gradient. After 4 hours, SW-13 cells expressing RASSF1A showed a 5-fold reduction in the number of cells migrated across the membrane (Figure 5B), suggesting a strong motility-inhibitory response from re-expressed RASSF1A. Similarly, cells constitutively expressing high levels of RASSF1A also showed a significantly impaired clonogenic survival/growth response when compared to cells not expressing RASSF1A or cells expressing high levels of RASSF1A/A133S mutant proteins (Figure 5C). In summary, RASSF1A expression resulted in an overall reduced malignant behavior of SW-13 ACC cells which was not observed in cells expressing the RASSF1A/A133S mutant protein.

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