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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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Co-localization of RASSF1A with microtubules. (A) SW-13/A (a, b &c) or SW-13/AM (d, e &f) cells were grown on glass cover slips in medium containing 400 ug/ml G418 and after 24 hours, cells were fixed in cold Acetone-Methanol (1:1) for 10 minutes followed by immunofluorescence detection of cytoskeleton (using Rhodamine-Phalloidin; a &d) or RASSF1A (using anti-RASSF1A antibody and FITC-conjugated secondary antibody; b &e) or both (c &f). Cell nuclei fluoresces blue with DAPI. Arrows indicate areas of co-localization of RASSF1A and cytoskeleton (c &f). (B) RASSF1A-microtubule co-localization points in comparable number of photomicrographs of SW-13/A and SW-13/AM cells representing multiple views from duplicate experiments were manually counted, tabulated and presented as a graph. (C) Representative photomicrographs showing indirect immunofluorescence detection of RASSF1A and microtubules in the normal adrenal cortex (a) and ACC (b) tissue specimens. Red fluorescence represents microtubules, green RASSF1A and blue DAPI-stained nuclei. Arrows indicate co-localization of RASSF1A and microtubules. (Total magnification: 1000X).
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Figure 6: Co-localization of RASSF1A with microtubules. (A) SW-13/A (a, b &c) or SW-13/AM (d, e &f) cells were grown on glass cover slips in medium containing 400 ug/ml G418 and after 24 hours, cells were fixed in cold Acetone-Methanol (1:1) for 10 minutes followed by immunofluorescence detection of cytoskeleton (using Rhodamine-Phalloidin; a &d) or RASSF1A (using anti-RASSF1A antibody and FITC-conjugated secondary antibody; b &e) or both (c &f). Cell nuclei fluoresces blue with DAPI. Arrows indicate areas of co-localization of RASSF1A and cytoskeleton (c &f). (B) RASSF1A-microtubule co-localization points in comparable number of photomicrographs of SW-13/A and SW-13/AM cells representing multiple views from duplicate experiments were manually counted, tabulated and presented as a graph. (C) Representative photomicrographs showing indirect immunofluorescence detection of RASSF1A and microtubules in the normal adrenal cortex (a) and ACC (b) tissue specimens. Red fluorescence represents microtubules, green RASSF1A and blue DAPI-stained nuclei. Arrows indicate co-localization of RASSF1A and microtubules. (Total magnification: 1000X).

Mentions: To test whether the observed malignant-dampening effect of RASSF1A in SW-13 cells is through modulating cytoskeletal function, we examined the localization pattern of RASSF1A (Figure 6A; b & c)) and RASSF1A/A133S mutant (Figure 6A; e & f) proteins in the context of localization of microtubule-binding phalloidins (Figure 6A; a, c, d & f). Co-localization of microtubules with RASSF1A was observed predominantly in cells expressing the wild-type RASSF1A protein (Note the arrows in Figure 6A; c), which was found significantly reduced (6B) in cells expressing RASSF1A/A133S mutant proteins (Figure 6A; f), suggesting a potential microtubule modulatory role for RASSF1A, not the mutant A133S mutant, in eliciting the observed reduced malignant behavior in SW-13 cells constitutively expressing RASSF1A. Despite the absence of RASSF1A/A133S co-localization with microtubules, the overall microtubule distribution appeared to be similar between RASSF1A-expressing and A133S mutant-expressing cells (6A; a & b). We also observed a similar co-localization pattern for RASSF1A and microtubules (Figure6C; a) in normal adrenal cortex where microtubules appeared to have a punctate co-localization pattern of distribution with RASSF1A, in comparison to a more dispersed distribution found in ACC specimens that lack RASSF1A expression (Figure 6C; b). Although indirect, the co-localization of RASSF1A with microtubules both in normal adrenal cortex and ACC cells with reduced malignant properties (SW-13/A) suggests an anti-motility role for RASSF1A in adrenocortical carcinogenesis.


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)

Co-localization of RASSF1A with microtubules. (A) SW-13/A (a, b &c) or SW-13/AM (d, e &f) cells were grown on glass cover slips in medium containing 400 ug/ml G418 and after 24 hours, cells were fixed in cold Acetone-Methanol (1:1) for 10 minutes followed by immunofluorescence detection of cytoskeleton (using Rhodamine-Phalloidin; a &d) or RASSF1A (using anti-RASSF1A antibody and FITC-conjugated secondary antibody; b &e) or both (c &f). Cell nuclei fluoresces blue with DAPI. Arrows indicate areas of co-localization of RASSF1A and cytoskeleton (c &f). (B) RASSF1A-microtubule co-localization points in comparable number of photomicrographs of SW-13/A and SW-13/AM cells representing multiple views from duplicate experiments were manually counted, tabulated and presented as a graph. (C) Representative photomicrographs showing indirect immunofluorescence detection of RASSF1A and microtubules in the normal adrenal cortex (a) and ACC (b) tissue specimens. Red fluorescence represents microtubules, green RASSF1A and blue DAPI-stained nuclei. Arrows indicate co-localization of RASSF1A and microtubules. (Total magnification: 1000X).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Co-localization of RASSF1A with microtubules. (A) SW-13/A (a, b &c) or SW-13/AM (d, e &f) cells were grown on glass cover slips in medium containing 400 ug/ml G418 and after 24 hours, cells were fixed in cold Acetone-Methanol (1:1) for 10 minutes followed by immunofluorescence detection of cytoskeleton (using Rhodamine-Phalloidin; a &d) or RASSF1A (using anti-RASSF1A antibody and FITC-conjugated secondary antibody; b &e) or both (c &f). Cell nuclei fluoresces blue with DAPI. Arrows indicate areas of co-localization of RASSF1A and cytoskeleton (c &f). (B) RASSF1A-microtubule co-localization points in comparable number of photomicrographs of SW-13/A and SW-13/AM cells representing multiple views from duplicate experiments were manually counted, tabulated and presented as a graph. (C) Representative photomicrographs showing indirect immunofluorescence detection of RASSF1A and microtubules in the normal adrenal cortex (a) and ACC (b) tissue specimens. Red fluorescence represents microtubules, green RASSF1A and blue DAPI-stained nuclei. Arrows indicate co-localization of RASSF1A and microtubules. (Total magnification: 1000X).
Mentions: To test whether the observed malignant-dampening effect of RASSF1A in SW-13 cells is through modulating cytoskeletal function, we examined the localization pattern of RASSF1A (Figure 6A; b & c)) and RASSF1A/A133S mutant (Figure 6A; e & f) proteins in the context of localization of microtubule-binding phalloidins (Figure 6A; a, c, d & f). Co-localization of microtubules with RASSF1A was observed predominantly in cells expressing the wild-type RASSF1A protein (Note the arrows in Figure 6A; c), which was found significantly reduced (6B) in cells expressing RASSF1A/A133S mutant proteins (Figure 6A; f), suggesting a potential microtubule modulatory role for RASSF1A, not the mutant A133S mutant, in eliciting the observed reduced malignant behavior in SW-13 cells constitutively expressing RASSF1A. Despite the absence of RASSF1A/A133S co-localization with microtubules, the overall microtubule distribution appeared to be similar between RASSF1A-expressing and A133S mutant-expressing cells (6A; a & b). We also observed a similar co-localization pattern for RASSF1A and microtubules (Figure6C; a) in normal adrenal cortex where microtubules appeared to have a punctate co-localization pattern of distribution with RASSF1A, in comparison to a more dispersed distribution found in ACC specimens that lack RASSF1A expression (Figure 6C; b). Although indirect, the co-localization of RASSF1A with microtubules both in normal adrenal cortex and ACC cells with reduced malignant properties (SW-13/A) suggests an anti-motility role for RASSF1A in adrenocortical carcinogenesis.

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