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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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Stable expression and selection of RASSF1A-expressing ACC cell lines. SW-13 ACC cells were transfected with CMV-promoted RASSF1A and RASSF1A/A133S mutant genes and G-418 resistant clones were selected under low-seeding densities. Multiple clones were pooled to make populations to avoid variability in expression levels. (A) Western Immunoblot detection of RASSF1A and RASSF1A/A133S (arrows point to the RASSF1A bands) expression using anti-DDK (a) or anti-RASSF1A (b) antibody (lanes a1: transfected with empty vector; lane a2: RASSF1A; lane a3: RASSF1A/A133S; b1 empty vector, and lane b2 RASSF1A expression vectors). (B) Immunofluorescence detection of RASSF1A expression in (a) SW-13/N, (b) SW-13/A, and (c) SW-13/M cells, using anti-RASSF1A mAb followed by anti-mouse-FITC antibody (green). DAPI stained nucleus appears blue. (C &D) Established populations were grown for 7 days to determine the effect of constitutive expression of RASSF1A or RASSF1A/A133S on proliferation (C) and survival (D) in comparison to vector-transfected and selected cells. Graphs represent data from one of two independent experiments with similar results.
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Figure 4: Stable expression and selection of RASSF1A-expressing ACC cell lines. SW-13 ACC cells were transfected with CMV-promoted RASSF1A and RASSF1A/A133S mutant genes and G-418 resistant clones were selected under low-seeding densities. Multiple clones were pooled to make populations to avoid variability in expression levels. (A) Western Immunoblot detection of RASSF1A and RASSF1A/A133S (arrows point to the RASSF1A bands) expression using anti-DDK (a) or anti-RASSF1A (b) antibody (lanes a1: transfected with empty vector; lane a2: RASSF1A; lane a3: RASSF1A/A133S; b1 empty vector, and lane b2 RASSF1A expression vectors). (B) Immunofluorescence detection of RASSF1A expression in (a) SW-13/N, (b) SW-13/A, and (c) SW-13/M cells, using anti-RASSF1A mAb followed by anti-mouse-FITC antibody (green). DAPI stained nucleus appears blue. (C &D) Established populations were grown for 7 days to determine the effect of constitutive expression of RASSF1A or RASSF1A/A133S on proliferation (C) and survival (D) in comparison to vector-transfected and selected cells. Graphs represent data from one of two independent experiments with similar results.

Mentions: The tumor suppressor function of RASSF1A is context-dependent and is elicited via multiple and alternate signaling events such as pro-apoptotic, cell cycle arrest, mitotic arrest and/or cytoskeletal modifications[15]. After confirming the lack of apoptosis-inducing and cell cycle arrest functions in SW-13 cells via transient transfection experiments (Figures 3B &3C), we generated stable SW-13 cell derivatives that expressed RASSF1A and RASSF1A/A133S mutant proteins, to study potential tumor suppressor functions of RASSF1A in adrenal carcinomas. Expression of RASSF1A (SW-13/A) and RASSF1A/A133S (SW-13/AM) was verified following neomycin selection and subsequent population expansion, using Western immunoblots (Figure 4A; lanes a2, a3 & b2) and immunofluorescence (Figure 4B). Wild-type RASSF1A were found distributed both in the cytoplasm and nucleus of cells (Figure 4B; b) while RASSF1A/A133S mutant proteins were found predominantly localized in the cytoplasm (Figure 4B; c). Note lack of expression of endogenous RASSF1A in SW-13/V cells (Figure 4A: lane b1 and Figure 4B; a). Similar to the observation in transiently transfected cells (Figure 3B &3C), no significant detrimental effects on cell viability or growth were observed following stable expression in SW-13/V, SW-13/A, or SW-13/AM populations (Figure 4C &4D), suggesting alternate roles for RASSF1A silencing in ACC and possibly in SW-13 cell behavior.


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)

Stable expression and selection of RASSF1A-expressing ACC cell lines. SW-13 ACC cells were transfected with CMV-promoted RASSF1A and RASSF1A/A133S mutant genes and G-418 resistant clones were selected under low-seeding densities. Multiple clones were pooled to make populations to avoid variability in expression levels. (A) Western Immunoblot detection of RASSF1A and RASSF1A/A133S (arrows point to the RASSF1A bands) expression using anti-DDK (a) or anti-RASSF1A (b) antibody (lanes a1: transfected with empty vector; lane a2: RASSF1A; lane a3: RASSF1A/A133S; b1 empty vector, and lane b2 RASSF1A expression vectors). (B) Immunofluorescence detection of RASSF1A expression in (a) SW-13/N, (b) SW-13/A, and (c) SW-13/M cells, using anti-RASSF1A mAb followed by anti-mouse-FITC antibody (green). DAPI stained nucleus appears blue. (C &D) Established populations were grown for 7 days to determine the effect of constitutive expression of RASSF1A or RASSF1A/A133S on proliferation (C) and survival (D) in comparison to vector-transfected and selected cells. Graphs represent data from one of two independent experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Stable expression and selection of RASSF1A-expressing ACC cell lines. SW-13 ACC cells were transfected with CMV-promoted RASSF1A and RASSF1A/A133S mutant genes and G-418 resistant clones were selected under low-seeding densities. Multiple clones were pooled to make populations to avoid variability in expression levels. (A) Western Immunoblot detection of RASSF1A and RASSF1A/A133S (arrows point to the RASSF1A bands) expression using anti-DDK (a) or anti-RASSF1A (b) antibody (lanes a1: transfected with empty vector; lane a2: RASSF1A; lane a3: RASSF1A/A133S; b1 empty vector, and lane b2 RASSF1A expression vectors). (B) Immunofluorescence detection of RASSF1A expression in (a) SW-13/N, (b) SW-13/A, and (c) SW-13/M cells, using anti-RASSF1A mAb followed by anti-mouse-FITC antibody (green). DAPI stained nucleus appears blue. (C &D) Established populations were grown for 7 days to determine the effect of constitutive expression of RASSF1A or RASSF1A/A133S on proliferation (C) and survival (D) in comparison to vector-transfected and selected cells. Graphs represent data from one of two independent experiments with similar results.
Mentions: The tumor suppressor function of RASSF1A is context-dependent and is elicited via multiple and alternate signaling events such as pro-apoptotic, cell cycle arrest, mitotic arrest and/or cytoskeletal modifications[15]. After confirming the lack of apoptosis-inducing and cell cycle arrest functions in SW-13 cells via transient transfection experiments (Figures 3B &3C), we generated stable SW-13 cell derivatives that expressed RASSF1A and RASSF1A/A133S mutant proteins, to study potential tumor suppressor functions of RASSF1A in adrenal carcinomas. Expression of RASSF1A (SW-13/A) and RASSF1A/A133S (SW-13/AM) was verified following neomycin selection and subsequent population expansion, using Western immunoblots (Figure 4A; lanes a2, a3 & b2) and immunofluorescence (Figure 4B). Wild-type RASSF1A were found distributed both in the cytoplasm and nucleus of cells (Figure 4B; b) while RASSF1A/A133S mutant proteins were found predominantly localized in the cytoplasm (Figure 4B; c). Note lack of expression of endogenous RASSF1A in SW-13/V cells (Figure 4A: lane b1 and Figure 4B; a). Similar to the observation in transiently transfected cells (Figure 3B &3C), no significant detrimental effects on cell viability or growth were observed following stable expression in SW-13/V, SW-13/A, or SW-13/AM populations (Figure 4C &4D), suggesting alternate roles for RASSF1A silencing in ACC and possibly in SW-13 cell behavior.

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