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

Article (PDF Available)inMolecular Cancer 12(1):87 · August 2013with32 Reads
DOI: 10.1186/1476-4598-12-87 · Source: PubMed
Abstract
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. 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. 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.
RES E A R C H Open Access
Epigenetic silencing of RASSF1A deregulates
cytoskeleton and promotes malignant behavior of
adrenocortical carcinoma
Reju Korah
1,2
, James M Healy
1,2
, John W Kunstman
1,2
, Annabelle L Fonseca
1,2
, Amir H Ameri
1
, Manju L Prasad
3
and Tobias Carling
1,2*
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.
Keywords: Adrenal cortex, Carcinoma, Adenoma, RASSF1A, Hypermethylation, Epigenetic silencing, Cytoskeleton
Background
Adrenocortical carcinoma (ACC) is a rare endocrine
malignancy, with an annual incidence of approximately
0.5 - 2 cases per million [1,2]. Despite rece nt progress,
including the first randomized c ontrolled trial of con-
ventional chemotherapy in ACC patients [3], these
tumors remain a clinical challenge, with an overall
5-year sur vival of 16-38% e ven with aggressive surgical
and oncologic therapy [4]. The major reasons for this
include (a) an initially silent clinical course that ultim-
ately manifests in advanced disease, with 30-40% of pa-
tients having metastatic disease upon initial diagnosis
[5,6] and, (b) the incomplete understanding of the mo-
lecular pathogenesis of the disea s e.
Several well-known tumor suppressor- and oncogene-
signaling pathways have been pre viously described as
implicated in ACC tumorigenesis. In addition to somatic
mutations in exons 58 and 211, germ-line variants in
* Correspondence: tobias.carling@yale.edu
1
Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University
School of Medicine, 333 Cedar Street, TMP202, Box 208062, New Haven, CT
06520, USA
2
Departments of Surgery, Yale University School of Medicine, New Haven, CT,
USA
Full list of author information is available at the end of the article
© 2013 Korah et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Korah et al. Molecular Cancer 2013, 12:87
http://www.molecular-cancer.com/content/12/1/87
TP53 also has been reported in sporadic ACCs [7-11].
Moreover, the Wnt signaling pathway is found fre-
quently altered in ACC, with abnormal accumulation of
β-catenin present in 85% of ACC, and somatic activ ating
mutations present in 30% of ACCs [12]. In patients with
Carney Complex and Isolated Primary Pigmented Nodu-
lar Adrenocortical Disease, somatic inactivating muta-
tions in PRKAR1A have been associated with ACC [13].
Although controversial, deregulated IGF signaling also
has been implicated in the origin and/or progression of
ACC [14].
The Ras-Associati on Domain Family 1A (RASSF1 A)
protein is a 37kDa ubiquitously exp res sed isoform
of the RASSF1 gene w ith demonstrated tumor
suppressor function in a variety o f tissu es [15-17].
RASSF1 is expressed a s multiple splice variants, with
each containing an RA domain, a C-terminal SARAH
protein-protein interaction motif, a phosphorylation
site for the DNA repa ir kinase ATM, and a cysteine-
rich domain homologous to the Raf-1 diacylglycerol
binding domain [15]. The RASSF1 gene has two asso ci-
ated CpG islands (CpG islands A & C), with the smaller
737 bp-CpG island A spanning the promoter region for
RASSF1A while CpG island C spans exon 2 that e n-
compasses promoter regions for isoforms R A SFF1B
and R A SFF1C [18].
Multiple studies have suggested a variety of roles for
RASSF1A in suppressing carcinogenesis. RASSF1A restricts
unscheduled proliferation, survival, and migration signaling
downstream of a variety of oncogenes, including RAS and
BRAF [19,20]. RASSF1A can regulate cell proliferation via
protein-protein interaction with RAS [21], can stabilize
microtubule formation via a domain near the ATM
phosphorylation targe t (S131) [22-25], and h a s demon-
strated pro-apoptotic effect s downstream to multiple
pathways including Hippo, SAPK-JNK,andMST1/MST2
[26]. RASSF1A can also suppress K-Ras and TNF-alpha
induced resistance to apoptosis [22,25,27,28]. Furthe r-
more, RASSF1A induction has been shown to suppress
anchorage-dependent colony formation in non-squamous
cell lung cancer cell-lines [29] and RASSF1A knockout
mice have increased susceptibility to spontaneous tumor
development [30]. However, its precise role and mechan-
ism of action in most tumor types remains to be further
clarified [31-34].
Epigenetic aberrations, including DNA methylation
and histone modifications, are increasingly being recog-
nized for their role in altering patterns of gene expres-
sion, potentially contributing to tumorigenesis [35].
Global DNA hypomethylation has been demonstrated in
ACC [36,37] with locus-specific patterns of hyper-
methylation [36,38]. Genome-wide studies of the DNA
methylomes of ACC and ACA have identif ied multiple
genes with differential DNA methylation patterns;
notably, including several tumor suppressor genes
[38,39].
Hypermethylation of the RASSF1 promoter respon-
sible for RA SSF1A expression has a well-established
role in tumor progression in se veral organ systems
and tissue types [26,40-45], including several endo-
crine tumors. Specifically, epigenetic suppression of
RASSF1A expression in papill ary thyroid carcinoma
has been strongly implicated in early tumor forma-
tion [31,32,46,47]. Similarly, epigenetic silencing of
RASSF1A has been demonstrated in neural crest tu-
mors such as neuroblastoma and pheochromocytoma
[46]. Alternatively, genetic silencing of RA SSF1A
gene by mutations and other aberrations are pos-
sible, but rarely seen in human cancers [47]. In this
study, we hypothesized that R ASSF1A functions as a
tumor suppressor in adrenal cortex and t hat it s epi-
genetic suppression by promoter methylation may be
a key step in tumor progression. W e a lso evaluated
whether RASSF1A suppression in ACC is correlated
with a more malignant phenotype. Furthermore, we
investigated the functional consequence of re versing
this suppression in an adrenocortical cell culture sys-
tem with the aim towards understanding the me ch-
anism of R ASSF1A function in the adrenal cortex.
Results
Increased hypermethylation of CpG island A of the
RASSF1 promoter in adrenocortical carcinoma
RASSF1 CpG island A hypermethylation is the most
common epigenetic mechanism observed in tumors
with silenced RA SSF1A function [31,32,46,47]. To test
whether promoter hypermethylation and consequent
RASSF1A silencing contributes to adrenocortical tu-
morigenesis, we first determined the methylation status
of CpG island A of RASSF1 in fresh-frozen ACC
(n = 7), ACA (adrenocortical adenoma; n = 8) and nor-
mal 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 methy-
lation. 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
Korah et al. Molecular Cancer 2013, 12:87 Page 2 of 13
http://www.molecular-cancer.com/content/12/1/87
normal and ACA cases (Figure 1B). Hypermethylation
in normal adrenal cortex samples ranged from 0.2 2.0
%withanaverageof0.35%andACAsamplesranged
from 0.03 1.7% with an average hypermethylation
of 0.7%. Conversely, all the ACC samples tested
showed hypermethylation in excess of the ma ximum
level observed in normal and ACA tissues. About
60% (4/7) of ACC samples had very high (>20%)
hypermethylation of the CpG island A o f the RASSF1
promoter (Figure 1B).
Reduced expression of RASSF1A in adrenocortical
carcinoma (ACC)
A direct correlation between RASSF1 prom oter hy-
permethylation and reduced RASSF1A expression is
observed in a variety of cancers (15). To test whether
the observed CpG Island A hypermethylation in ACC
(Figure 1B) is associated with a corresp onding reduction
in RASSF1A expression, we compared the mRNA and
protein expression in ACC samples to that of normal ad-
renal cortex tissue samples. Quantitative PCR analysis of
AB
CD
a
b
c
d
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).
Korah et al. Molecular Cancer 2013, 12:87 Page 3 of 13
http://www.molecular-cancer.com/content/12/1/87
gene expression showed significantly decreased expression
of RASSF1A mRNA in ACC samples (P < 0.01) ranging
from 1% to 24% of average RASSF1A expression in nor-
mal adrenal cortex (Figure 1C). All ACC samples tested
showed reduced expression of RASSF1A, irrespective of
their clinical characteristics or malignant stages status
(Table 1; Figure 1C). We also assessed the expression
levels of RASSF1A protein in ACC samples by immuno-
histochemical staining. Histopathologically normal speci-
mens (Figure 1D; a & b) showed markedly higher
RASSF1A expression, while RASSF1A was undetectable
in areas dominated by malignant cells (Figure 1D; c & d),
suggesting a correlation between low mRNA expression
and undetectable RASSF1A protein levels in ACC
samples.
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 im-
munofluorescence (Figure 2A). As shown in Figure 2A,
both ACC cell lines revealed undetectable levels of
RASSF1A, when compared with the expression in a thy-
roid 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 show ed 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 pro-
moter 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).
Enforced expression of RASSF1A in SW-13 cells
To test whether RASSF1A silencing contrib utes to
the malignant progression of ACC, we re-expressed
RASSF1A in SW-13 ACC cells that did not express de-
tectable levels of RASSF1A (Figure 2A). In addition to
the empty ve ctor transfection controls (Figure 3A; a, e),
we also expressed a variant of RASSF1A (RASSF1A/
A133S) with demonstrated inability to elicit tumor
suppressor function [48], as a control. Expression of ec-
topic RASSF1A (Figure 3A; b-d) and RASSF1A/A133S
(Figure 3A; f-h) were confirmed using anti-RASSF1A
antibody (Figure 3A; b & f) or anti-DDK antibody
(Figure 3A; c & g), both of which co-localized to the
same epitope (Figure 3A; d & h). After confirming the
transfection efficiency to be in excess of 70% (Figure 3A)
one day post-transfection, we determined the cell prolif-
eration efficiency and cell survival for a period of 6 days
post-transfection. Constitutive expression of RASSF1A
(SW-13/A) or RASSF1A/A133S mutant (SW-13/AM)
showed no sign ific ant impact on the proliferation
(Figure 3B) or viability (Figure 3C) of SW-13 cells in
comparison with SW-13 transiently transfected with
the empty vector (SW-13/V) alone (Figures 3B & 3C).
The tumor suppressor function of RASSF1A is
context-dependent and is elicited via multiple and alter-
nate signaling events such as pro-apoptotic, cell cycle ar-
rest, mitotic arrest and/or cytoskeletal modifications
[15]. After confirming the lack of apoptosis-inducing
and cell cycle arrest functions in SW-13 cells via transi-
ent 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 fol-
lowing neomycin selection and subsequent population
expansion, using Western immunoblots (Figure 4A;
Table 1 Clinicopathological characteristics of patients
ID Age Gender ENSAT 2008 Stage Metastasis Diameter Hormonal profile Recurrence
C1 58 F III N 13 cm Non-functional Y
C2 32 F III N 12 cm Androgen-producing N
C3 62 F III Y 14 cm Cortisol-producing Y
C4 48 M III N 9 cm Non-functional N
C5 48 F III N 13 cm Cortisol-producing Y
C6 55 F IV Y 5.5 cm Cortisol-producing Y
C7 60 F IV Y 7.8 cm Cortisol-producing Y
Diverse clinical and pathological characteristics of adrenocortical cancer (n = 7) patients selected for the study, is shown. ENSAT 2008 Staging: European Network
for the Study of Endocrine Tumors; Y = Yes, N = No, F = Female, M = Male.
Korah et al. Molecular Cancer 2013, 12:87 Page 4 of 13
http://www.molecular-cancer.com/content/12/1/87
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 predom-
inantly 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 obser ved following stable expression
in SW-13/V, SW- 13/A, or SW-13/A M popu lations
(Figure 4C & 4D), suggesting alternate roles for RASSF1A
silencing in ACC and possibly in SW-13 cell behavior.
Re-expression of RASSF1A reduces malignant behavior of
SW-13 cells
Advanced features of malignancy, such as invasion of or
migration to adjacent tissues, degradation of the extra
AB
CD
0
10
20
30
40
50
60
70
80
90
100
NCI-H295R SW-13
Percent Methylation of RASSF1A Promoter
FHM
FIM
FUM
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
00.11 5 10
Percent Methylation of RASSF1A Promoter
5-aza-2'-deoxycytidine (µM)
FIM
FHM
FUM
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
00.11 5 10
RASSF1A Expression Normalized to Untreated
Control
5-aza-2'-deoxycytidine (µM)
ACT-1
NCI-H295R
SW-13
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.
Korah et al. Molecular Cancer 2013, 12:87 Page 5 of 13
http://www.molecular-cancer.com/content/12/1/87
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 poten-
tial 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. Simi-
larly, cells constitutively expressing high levels of
RASSF1A also showed a significantly impaired clonogenic
sur vival/growth response when compared to cells not
expressing R A SSF1A or cells expressing high le vels
of RASSF1A/A133S mutant proteins (Figure 5 C) . In
summary, RASSF1A expression resulte d in an overall
A
BC
0
25
50
75
100
125
150
0123456
Number of cells X 10
4
Number of Days in Culture
SW-13/V
SW-13/A
SW-13/AM
-10
0
10
20
30
40
50
60
70
80
90
100
0123456
Percecent Non-viable Cells
Number of Days in Culture
SW-13/V
SW-13/A
SW-13/AM
Figure 3 Enforced expression of RASSF1A in ACC cells. (A) SW-13 ACC cells that do not express RASSF1A (a) were transfected with empty (a
& e), RASSF1A (b - d) or RASSF1A/A133S (f - h) expression vectors. Expression of RASSF1A was determined by immunofluorescence detection of
RASSF1A protein (b & f) or DDK tag (c & g) using anti-RASSF1 goat polyclonal and anti-DDK mAb respectively followed by anti-goat-FITC and
anti-moue-TR secondary antibodies and DAPI for nuclear staining. Note co-localization of RASSF1A and DDK antigens (d & h) and absence of
both in a & e. (B & C) Transient transfection was carried out in 6-well plates with a starting density of 80,000 cells/well and allowed to grow for 6
days to test the effect of RASSF1A and RASSF1A/A133S expression on growth potential (B) and survival (C) of SW-13 cells. Graphs represent one
of 3 independent experiments that yielded similar results.
Korah et al. Molecular Cancer 2013, 12:87 Page 6 of 13
http://www.molecular-cancer.com/content/12/1/87
reduced malignant b ehavior of SW-13 ACC cells which
was not observed in cells expressing the R ASSF1A/
A133S mutant protein.
RASSF1A alters malignant behavior of ACC cells by
modulating microtubule organization
To test whether the observed malignant-dampening ef-
fect of RASSF1A in SW-13 cells is through modulating
cytoskeletal function, we examined the localization pat-
tern 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 ex-
pressing the wild-type RASSF1A protein (Note the ar-
rows in Figure 6A; c), which was found significantly
reduced (6B) in cells expressing RASSF1A/A133S mu-
tant proteins (Figure 6A; f ), suggesting a potential
microtubule modulatory role for RASSF1A, not the mu-
tant A133S mutant, in eliciting the observed reduced
malignant behavior in SW-13 cells constitutively ex-
pressing RASSF1A. Despite the absence of RASSF1A/
A133S co-localization with microtubules, the overall
microtubule distribution appeared to be similar between
A
B
CD
0
10
20
30
40
50
0123456
Number of Cells X 10
4
Days in Culture
SW-13/V
SW-13/A
SW-13/AM
-10
0
10
20
30
40
50
60
70
80
90
100
0123456
Percentage Non-viable Cells
Days in Culture
SW-13/V
SW-13/A
SW-13/AM
b
a
123
1 2
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.
Korah et al. Molecular Cancer 2013, 12:87 Page 7 of 13
http://www.molecular-cancer.com/content/12/1/87
RASSF1A-expressing and A133S mutant-expressing
cells (6A; a & b). We also obser ved a similar co-
localization pattern for R A SSF1A and microtubules
(Figure 6C; a) in normal adrenal cortex where microtu-
bules 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 malig-
nant properties (SW-13/A) suggests an anti-motility role
for RASSF1A in adrenocortical carcinogenesis.
Discussion
Neoplasias of the adrenal cortex present unique chal-
lenges in diagnosis and treatment, largely due to an in-
complete understanding of the molecular pathogenesis
of the disease. In this study, we examin e the role of
RASSF1A, a well-known tumor suppressor that ha s
demonstrated roles in numerous other malignancies
including several endocrine cancers, in adrenocortical
carcinogenesis. Recent studies in gene expression profil-
ing have suggested a potential role for aberrant DNA
methylation events (both hypo- and hypermethylation)
in the origin and/or progression of ACC, as in many
other malignancies, including endocrine tumors such as
neuroblastoma and pheochromocytoma [35-38,46,49,50].
RASSF1A, the most frequently silenced tumor suppres-
sor via promoter methylation [15], thus is an attractive
candidate to explore in the context of adrenocortical
tumorigenesis.
Interrogation of the CpG Island A of the RASSF1 pro-
moter using the methyl screen technology showed a
markedly increased hypermethylation pattern in ACC
tissue samples. The promoter hypermethylation pattern
observed in ACC was distinctly different from both nor-
mal adrenal cortex samples and benign adenomas,
suggesting RASSF1A silencing as a possible later event
in the overall adrenocortical tumorigenesis process. Ex-
pression analysis of RASSF1A in ACC demonstrated the
ABC
0
50
100
150
200
250
300
350
400
450
500
SW-13/V SW-13/A SW-13/AM
Number of Cells Invaded Through Matrigel
0
200
400
600
800
1000
1200
SW-13/V SW-13/A SW-13/AM
Number of clones with 10 +/-2 cells
p= <0.01
0
20
40
60
80
100
120
SW-13/V SW-13/A SW-13/AM
Number of Cells Migrated in 4 Hours
p= <0.001
p= <0.001
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.
Korah et al. Molecular Cancer 2013, 12:87 Page 8 of 13
http://www.molecular-cancer.com/content/12/1/87
functional consequence of hypermethylation as a signifi-
cant decrease in both gene transcription and translation
of RASSF1A in ACC cells as determined by qPCR, and
immunohistochemistry respectively.
To test the hypothesized role for RASSF1A silencing
in promoting adrenocortical malignancy, we sought to
use a well-established cell culture model. We chose SW-
13 ACC cell line that showed a comparable RASSF1 pro-
moter CpG Island A hypermethylation and undetectable
RASSF1A protein expression. As the tumor suppressor
function of RASSF1A manifests in promotion of
apoptosis and downregulation of cell proliferation, via-
bility and proliferation of SW-13 cells was first assessed
following transient transfection with RASSF1A-expressing
vector and found to be unaffected. Lack of promotion
of cell death or cell cycle arrest by R A SSF1A re-
expression allowed us to generate SW-13 cell derivatives
constitutively expressing RASSF1A. After confirming no
growth disadvantage consequent to enforced over expres-
sion of RASSF1A, SW-13 cell variants constitutively
expressing RASSF1A and RASSF1A/A133S were then
assayed for other advanced malignancy or metastasis
A
BC
a
b
a
bc
dfe
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).
Korah et al. Molecular Cancer 2013, 12:87 Page 9 of 13
http://www.molecular-cancer.com/content/12/1/87
associated tumor behaviors such as invasion and solitary
cell growth. Using a reconstituted basement membrane
(Matrigel) invasion assay, a significant reduction in inva-
siveness was observed in SW-13 cells with forced
RASSF1A expression (SW-13/A). To test whether the re-
duced invasive potential we observed in SW-13 cells with
forced RASSF1A expression was due to decreased migra-
tory ability, transwell migration assays were performed.
The results mirrored the invasion assay, demonstrating sig-
nificantly lower migratory potential with forced wild-type
RASSF1A expression that was absent in SW-13/AM. The
RASSF1A-mediated inhibition of the migratory response
appeared to be sufficient to account for the observed anti-
invasive effect and therefore, we did not investigate poten-
tial involvement of matrix metalloproteinases (MMPs)
or their inhibitors in mediating the observed Matrigel
invasion-inhibitory response. Finally, under low seeding
conditions that mimic solitary cell growth, constitutive ex-
pression of RASSF1A reduced the clonogenicity of solitary
SW-13 cells with decreased ability to establish, survive
and grow into individual clones. The solitary growth in-
hibitory effect was not found in RASSF1A/A133S-
expressing SW-13 cells.
Changes in microtubule dynamics are essential events
in initial local tumor invasion as well as later metastatic
spread. RASSF1A has demonstrated ability in influencing
cytoskeletal dynamics by physically binding to filamentous
actin as well as inhibiting tubulin polymerization
[23,24,28,51,52]. To test whether ectopically-expressed
RASSF1A physically interacts with microtubules, we
treated cells with microtubule-binding phalloidin along
with RASSF1A-detecting antibodies. In cells expressing
RASSF1A, but not in cells expressing the A133S mutant,
we observed sporadic co-localization of RASSF1A with
microtubules, which may have a stabilizing effect on
microtubule dynamics. Interestingly, it has been recently
suggested that the A133S point mutation in RASSF1A ab-
rogates its ability to modulate cytoskeletal interactions,
contributing to loss of its tumor suppressor function
[53]. Stabilization of microtubules by RASSF1A has
been shown to disrupt malignant behavior in many
cancer cell types [54]. Although RASSF1A-microtubule
co-localization was not ubiquitously obser ved throughout
the cell, the detected limited interaction may lead to
partial global disruption of microtubule dynamics and
contribute to the observed dampening of malignant be-
haviors in SW-13 cells. To assess whether the implicated
cytoskeleton-stabilizing role is absent in ACC tissue, we
compared the general organization of cytoskeleton in
ACC tissue to that of normal adrenal cortex. In ACC,
the cytoskeleton appeared to be very diffuse while
in the normal cortex, the cytoskeleton co-localized
with RA SSF1A into a pattern of punctuated struc tures
(Figure 6C), suggestive of a more stabilized organization.
However, more experiments are needed to confirm
the functional significance of such predicted RASSF1A-
cytoskeletal interactions.
In summary, the res ults of this study strongly suggest
functional evidence of a potential onc osuppressor role
for RASSF1A in adrenocortical carcinogenesis. Although
implicated to play a cytoskeleton-modulating role in
other tissues, this study provides the first evidence for a
cytoskeleton-stabilizing role for RASSF1A in adrenal
cortex. Whether silencing of RASSF1A serves as a driv-
ing event driving benign ACAs to malignant ACC status,
need further investigation.
Materials and methods
Tissue acquisition
Informed consent was obtained from patients prior to
surgical resection of adrenal tissue according to a proto-
col approved by the local Institutional Review Board and
Yale Pathology Tissue ser vices. Tissue was flash-frozen
in liquid nitrogen and stored at -80°C until processed for
study. Specimens displaying unequivocal histopatho-
logical characteristics of ACC (n = 7), ACA (n = 8), and
normal adrenal cortical tissue (n = 6) samples were se-
lected for use in the study. Consecutive unstained and
Hematoxylin & Eosin (H&E) stained 5 μm sections of
tumor and normal formalin-fixed paraffin embedded
(FFPE) tissue samples were obtained from Yale Tissue
Pathology services. All samples were evaluated by expe-
rienced endocrine pathologists before processing.
DNA, RNA, and Protein preparation
Genomic DNA from tissue samples were isolated using
the DNeasy blood and tissue kit from Qiagen (Valencia ,
CA). Total RNA from the samples were isolated using
the RNeasy Mini Kit (Qiagen, Valencia, CA,) after rotor-
stator homogenization, per the manufacturers recom-
mendations. Quantity and quality of prepared DNA and
RNA was assessed by spectrophotometry (NanoDrop
Technologies, Inc., Thermo Fischer, Waltham, MA) and
1% agarose gel electrophoresis. Total protein from cul-
tured cells were extracted using L aemmli buffer
(BioRad, Herc ules, CA) and protein concentrations
were measured using the Pierce B CA Protein assay Kit
(Thermo Scientific, Rockford, IL) and Multimax dete c-
tion system (Promega , Madison, WI), per the manufac-
turersinstructions.
Gene expression analysis
Total RNA (100 ng) was reverse transcribed using Super-
script III reverse transcriptase (Applied Biosystems,
Rockville, MD). Quantitative real-time PCR (qPCR) was
performed on triplicate samples using TaqMan PCR mas-
ter mix with the FAM flurophore and probe/primer pairs
specific to RASSF1A (Applied Biosystems, Rockville, MD)
Korah et al. Molecular Cancer 2013, 12:87 Page 10 of 13
http://www.molecular-cancer.com/content/12/1/87
according to the manufacturers cycling conditions using
CFX96 thermal cyclers (Bio-Rad, Hercules, CA). Gene ex-
pression levels were normalized to the averages of expres-
sion levels of beta-actin and TATA-binding protein probe/
primer pairs (Applied Biosystems, Rockville, MD). The
Cycle Threshold (C
T
) values were calculated using the
recommended Livak method (Bio-Rad, Hercules, CA).
Methylation-specific PCR
Methylation status of CpG Island A of the RASSF1A
promoter was assessed by MethylScreen technology
using the Epitect methyl II PCR assay (Qiagen, Valencia,
CA). Briefly, 125 ng of genomic DNA was mock-
digested or digested with methylation-sensitive and
methylation-dependent restriction enzymes individually
or together, and the methylation status of the target se-
quence was measured using real-time qPCR with probes
specific to the targ et promoter sequences. The amplifica-
tion results that corresponds to >60% digestion by
methylation-dependent restriction enzyme represents
Hypermethylated sequences and 0% digestion indicates
completely unmethylated DNA. Any amount of diges-
tion between 0% and 60% re present s the intermediate
methylation fraction. The Cycle Threshold (C
T
)values
were converted into percentages of unmethylated,
intermediately-methylated and hypermethylated CpG
values, using a quantitation algorithm provided by the
manufacturer (EpiTect Methyl II PCR Assay Handbook
Qiagen, Valencia, CA).
Immunohistochemical (IHC) and Immunofluorescence (IF)
detection
Five μm-thick FFPE sections were processed for immuno-
histochemistry according to the protocol recommended
by the manufacturer of 3,3Diaminobenzidine (DAB)
substrate (BD Biosciences, San Jose, CA). Mouse anti-
RASSF1A (1:100) primary antibody (Abcam, Cambridge,
MA), goat anti-mouse/Biotin antibody (Santa Cruz
Biotech., Santa Cruz, CA) and streptavidin-HRP (Life
technologies, Rockville, MD) were used prior to DAB sub-
strate development and detection (BD Biosciences, San
Jose, CA). Nikon Eclipse E600 microscope with Spot
3.5 program was used to take photomicrographs at a total
magnification of 400X. Immunofluorescence detection of
RASSF1A proteins and microtubules were carried out as
described [55]. Mouse anti-RASSF1A mAb (1:100;
Abcam, Cambridge, MA) or goat anti-RASSF1A goat
polyclonal (1:200; Santa CruZ Biotech, Santa Cruz, CA)
primary antibodies and anti-goat FITC, anti-goat TR, anti-
mouse FITC, or anti-mouse TR secondary antibodies
(1:1000; all from Santa Cruz Biotech., Santa Cruz, CA)
were used followed by ultracruz mounting agent
containing 4,6-diamidino-2-phenylindole - DAPI (Santa
Cruz Biotech., Santa Cruz , CA) for immunodetection.
Anti-DDK (DYKDDDDK epitope) monoclonal antibody
(1:200; Origene, Rockville, MD) was use d for dete cting
DDK - tagged RASSF1A and RASSF1A/A133S in transfected
cells. Rhodamine Phalloidin, a high affinity F-actin
probe conjugated to the red-orange fluorescent dye,
tetramethylrhodamine (TRITC) (Biotium, Hayward,
CA) was used for immunofluorescence detection of mi-
crotubules. Dilutions and incubations were ca rried out
per the manufacturers recommendations. Zeiss AX10
confocal microscope with AxioVision 4.8 program was
used for immunofluorescence analysis and photomicro-
graphs were taken at a total magnification of 1000 X.
Cell culture, expression vectors, transfections, and
Western blot detection
The human ACC cell line SW-13 was purchased from
American Type Cell Collection (Manassas, VA) and
was maintained under sterile conditions in DMEM
supplemented with 10% certified fetal bovine serum
and 10,000 U/mL penicillin/streptomycin (all from Life
Technologies, Inc., Rockville, MD) in a standard humidified
incubator at 37.0 C and 5% CO
2
. Myc-DDK tagged
pCMV6-Entry, pCMV6-Entry/RASSF1A, and pCMC6-
Entry/RASSF1A/A133S plasmid vectors (Origene, Rockville,
MD) were used for transfection. Transfected SW-13 cells
were designated SW-13/V representing pCMV vector alone,
SW-13/A representing pCMV-RASSF1A, and SW-13/AM
representing pCMV-RASSF1A/A133S mutant.
Transient transfection was carried out using Lipo-
fectamine2000 according to the manufacturers recom-
mendations (Life Technologies, Inc., Rockville, MD) in
6-well plates with a starting density of 80,000 cells/well.
Transfected cells were allowed to grow for 6 days, to test
the effect of RASSF1A and RA SSF1A/A133S mutant
gene expression on growth potential and sur vival of
SW-13 cells. Total cell numbers and viability were
calculated b y staining cells with 0.4% Trypan Blue
(GIBCO-BRL,LifeTechnologies,Inc.,Rockville,MD)
and manual counting using a counting chamber (Housser
Scientific Co., PA). Experiments were performed in tripli-
cate, and parallel plates with cells growing on glass cover-
slips were used to determine transfection efficiency and
continued expression of transfected genes by indirect
immunofluorescence.
Stable clones expressing RASSF1A and RASSF1A/
A133S were selected in 800 μg/ml G-418 (Life technolo-
gies Inc., Rockville, MD) containing growth medium.
Multiple clones were then pooled into populations to
avoid expression variability between clones. Established
populations (designated SW-13/V, SW-13/A, and SW-13
/AM representing pCMV vector alone, pCMV-RASSF1A,
and pCMV-RASSF1A/A133S mutant, respectively) were
used to determine the effects of constitutive expression of
RASSF1A or RASSF1A/A133S on SW-13 cells malignant
Korah et al. Molecular Cancer 2013, 12:87 Page 11 of 13
http://www.molecular-cancer.com/content/12/1/87
behavior. Expression of transfected genes were confirmed
via Western blotting using anti-DDK mAb for (1:1000;
Origene, MD), anti-RASSF1A mAb (1:500, Abcam, MA),
anti-mouse-HRP (Santa Cruz Biotech., CA), mini-
PROTEAN TGX gel, PVDF blotting membrane (BioRad,
Hercules, CA), and enhanced chemiluminescnce (ECL)
detection reagents (Pierce Thermo Scientific, Rockford,
IL) according to the manufacturers protocols. Equal pro-
tein loading between lanes were confirmed by staining
PVDF membranes after chemiluminescence detection.
Cell migration, invasion, and clonogenicity assays
Stable SW-13/V, SW-13/A or SW-13/AM cells were
allowed to invade through a Matrigel layer from upper
chambers containing serum-free medium to the lower
chamber containing 10% FBS medium in BDBiocoat
matrigel invasion chambers (BD Biosciences, Bedford,
MA). After 24 hours, the Matrigel was removed, and in-
vaded cells were fixed in 3.7% formaldehyde/PBS for 10
minutes, stained with 0.5% crystal violet for 2 hours, and
counted using 10X magnification with a light micro-
scope. The Matrigel invasion assay was performed twice
in duplicate chambers. In the migration assay, the
stably-transfected cells were allowed to migrate through
8 uM pore size modified Boyden Chambers (BD Biosci-
ences, Bedford, MA) from upper chambers containing
serum free medium to the lower chamber containing
10% FBS medium. After 4 hours, cells that migrated to
the lower side of the membrane towards a higher FBS
concentration gradient were fixed in 3.7% formaldehyde/
PBS for 10 minutes, stained with 0.5% crystal violet and
tabulated in triplicate. For clonogenicity assays, the cells
were seeded in 6-well plates in low densities (5000 cells/
well) and allowed to grow for 7 days in 400 μg/ml G-418
containing growth medium with a change of medium
after 3 days. Cells wer e washed with PBS, fixed with
3.7% formaldehyde/PBS solution, stained with 0.5% crys-
tal violet, and colonies with 10 +/ 2 cells were counted
and averaged from 6 wells after performing the assay in
quadruplicate.
Statistical analysis
Significance of observed differences in sample means
was evaluated using independent samples t-tests or
ANOVA where appropriate after ensuring normality
of distribution (Shapiro-Wilk test) and equivalence
of variance (Le venestest).P-values less than 0.05 were
considered to be significant in all cases. Analysis
was p erformed using SPSS v.19 (IBM Corporation,
Armonk, NY ).
Competing interests
The authors declare that there are no competing interests that could be
perceived as prejudicing the impartiality of the reported study.
Authors contributions
T Carling and R Korah conceived, developed, designed and coordinated the
study. R Korah performed gene expression studies, IF and IHC. J Healy and
J W Kun stman conducted biochemical assays, statistical analyses, provided
intellectual input and participated in the preparation of the manuscript. A
Ameri participated in the cell culture work. A Fonseca participated in the
procurement and processing of fresh-frozen and FFPE specimens. M Prasad
provided histopathological expertise and critically revised the manuscript.
All authors read and approved the final manuscript.
Grant support
This work was supported by an Ohse Research Award. T.C. is a Doris Duke-
Damon Runyon Clinical Investigator supported in part by the Damon
Runyon Cancer Research Foundation and the Doris Duke Charitable
Foundation.
Author details
1
Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University
School of Medicine, 333 Cedar Street, TMP202, Box 208062, New Haven, CT
06520, USA.
2
Departments of Surgery, Yale University School of Medicine,
New Haven, CT, USA.
3
Department of Pathology, Yale University School of
Medicine, New Haven, CT, USA.
Received: 8 May 2013 Accepted: 3 August 2013
Published: 5 August 2013
References
1. Wajchenberg BL, Albergaria Pereira MA, Medonca BB, Latronico AC, Campos
Carneiro P, Alves VA, et al: Adrenocortical carcinoma: clinical and
laboratory observations. Cancer 2000, 88:71136.
2. Lehmann T, Wrzesinski T: The molecular basis of adrenocortical cancer.
Cancer Genet 2012, 205:1317.
3. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, et al:
Combination chemotherapy in advanced adrenocortical carcinoma.
N Engl J Med 2012, 366:218997.
4. Berthon A, Martinez A, Bertherat J, Val P: Wnt/beta-catenin signalling in
adrenal physiology and tumour development. Mol Cell Endocrinol 2012,
351:8795.
5. Dackiw AP, Lee JE, Gagel RF, Evans DB: Adrenal cortical carcinoma. World
J Surg 2001, 25:91426.
6. Ng L, Libertino JM: Adrenocortical carcinoma: diagnosis, evaluation and
treatment. J Urol 2003, 169:511.
7. Ohgaki H, Kleihues P, Heitz PU: p53 mutations in sporadic adrenocortical
tumors. Int j cancer J int du cancer 1993, 54:40810.
8. Reincke M, Karl M, Travis WH, Mastorakos G, Allolio B, Linehan HM, et al: p53
mutations in human adrenocortical neoplasms: immunohistochemical
and molecular studies. J clin endocrinol metab 1994, 78:7904.
9. Wagner J, Portwine C, Rabin K, Leclerc JM, Narod SA, Malkin D: High
frequency of germline p53 mutations in childhood adrenocortical
cancer. J Natl Cancer Inst 1994, 86:170710.
10. Libe R, Groussin L, Tissier F, Elie C, Rene-Corail F, Fratticci A, et al: Somatic
TP53 mutations are relatively rare among adrenocortical cancers with
the frequent 17p13 loss of heterozygosity. Clin cancer res off j Am Assoc
Cancer Res 2007, 13:84450.
11. Soon PS, McDonald KL, Robinson BG, Sidhu SB: Molecular markers and the
pathogenesis of adrenocortical cancer. Oncologist 2008, 13:54861.
12. Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagnere AM, et al:
Mutations of beta-catenin in adrenocortical tumors: activation of the
Wnt signaling pathway is a frequent event in both benign and
malignant adrenocortical tumors. Cancer res 2005, 65:76227.
13. Bertherat J, Groussin L, Sandrini F, Matyakhina L, Bei T, Stergiopoulos S, et al:
Molecular and functional analysis of PRKAR1A and its locus (17q22-24)
in sporadic adrenocortical tumors: 17q losses, somatic mutations, and
protein kinase A expression and activity. Cancer res 2003, 63:530819.
14. Heaton JH, Wood MA, Kim AC, Lima LO, Barlaskar FM, et al: Progression to
adrenocortical tumorigenesis in mice and humans through insulin-like
growth factor 2 and b-catenin. Am J Pathol 2012, 181(3):101733.
15. Donninger H, Vos MD, Clark GJ: The RASSF1A tumor suppressor. J Cell Sci
2007, 120:3163 72.
16. Avruch J, Xavier R, Bardeesy N, Zhang XF, Praskova M, Zhou D, et al: Rassf
family of tumor suppressor polypeptides. J Biol Chem 2009, 284:110015.
Korah et al. Molecular Cancer 2013, 12:87 Page 12 of 13
http://www.molecular-cancer.com/content/12/1/87
17. Fausti F, Di Agostino S, Sacconi A, Strano S, Blandino G: Hippo and rassf1a
Pathways: A Growing Affair. Mol biol int 2012, 2012:307628.
18. Agathanggelou A, Cooper WN, Latif F: Role of the Ras-association domain
family 1 tumor suppressor gene in human cancers. Cancer Res 2005,
65:34973508.
19. Richter AM, Pfeifer GP, Dammann RH: The RASSF proteins in cancer; from
epigenetic silencing to functional characterization. Biochim Biophys Acta
2009, 1796:114 28.
20. Dumitrescu RG: Epigenetic markers of early tumor development.
Methods Mol Biol 2012, 863:314.
21. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D: RAS oncogenes: weaving a
tumorigenic web. Nature rev Cancer 2011, 11:76174.
22. Song MS, Song SJ, Ayad NG, Chang JS, Lee JH, Hong HK, et al: The tumour
suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20
complex. Nat Cell Biol 2004, 6:12937.
23. Liu L, Tommasi S, Lee DH, Dammann R, Pfeifer GP: Control of microtubule
stability by the RASSF1A tumor suppressor. Oncogene 2003, 22:812536.
24. Dallol A, Agathanggelou A, Fenton SL, Ahmed-Choudhury J, Hesson L, Vos
MD, et al: RASSF1A interacts with microtubule-associated proteins and
modulates microtubule dynamics. Cancer Res 2004, 64:41126.
25. Vos MD, Ellis CA, Bell A, Birrer MJ, Clark GJ: Ras uses the novel tumor
suppressor RASSF1 as an effector to mediate apoptosis. J Biol Chem 2000,
275:3566972.
26. Fernandes MS, Carneiro F, Oliveira C, Seruca R: Colorectal cancer and
RASSF family-A special emphasis on RASSF1A. Int J Cancer 2012,
132(2):2518.
27. Baksh S, Tommasi S, Fenton S, Yu VC, Martins LM, Pfeifer GP, et al: The
tumor suppressor RASSF1A and MAP-1 link death receptor signaling to
Bax conformational change and cell death. Mol Cell
2005, 18:63750.
28. Dallol A, Agathanggelou A, Tommasi S, Pfeifer GP, Maher ER, Latif F:
Involvement of the RASSF1A tumor suppressor gene in controlling cell
migration. Cancer Res 2005, 65:76539.
29. Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B,
et al : Epigenetic inactivation of RASSF1A in lung and breast cancers and
malignant phenotype suppression. J Natl Cancer Inst 2001, 93:6919.
30. Tommasi S, Dammann R, Zhang Z, Wang Y, Liu L, Tsark WM, et al: Tumor
susceptibility of Rassf1a knockout mice. Cancer Res 2005, 65:928.
31. Schagdarsurengin U, Gimm O, Hoang-Vu C, Dralle H, Pfeifer GP, Dammann
R: Frequent epigenetic silencing of the CpG island promoter of RASSF1A
in thyroid carcinoma. Cancer Res 2002, 62:3698701.
32. Xing M, Cohen Y, Mambo E, Tallini G, Udelsman R, Ladenson PW, et al: Early
occurrence of RASSF1A hypermethylation and its mutual exclusion with
BRAF mutation in thyroid tumorigenesis. Cancer Res 2004, 64:16648.
33. Lee SJ, Lee MH, Kim DW, Lee S, Huang SM, Ryu MJ, et al: Cross-Regulation
between Oncogenic BRAF(V600E) Kinase and the MST1 Pathway in
Papillary Thyroid Carcinoma. Plos One 2011, 6(1):e16180.
34. Catalano MG, Fortunati N, Boccuzzi G: Epigenetics modifications and
therapeutic prospects in human thyroid cancer. Front Endocrinol
(Lausanne) 2012, 3:40.
35. Fonseca AL, Kugelberg J, Starker LF, Scholl U, Choi M, Hellman P, et al:
Comprehensive DNA methylation analysis of benign and malignant
adrenocortical tumors. Genes Chromosomes Cancer 2012, 51:94960.
36. Jain M, Rechache N, Kebebew E: Molecular markers of adrenocortical
tumors. J Surg Oncol 2012, 106:54956.
37. Rechache NS, Wang Y, Stevenson HS, Killian JK, Edelman DC, Merino M,
et al : DNA methylation profiling identifies global methylation differences
and markers of adrenocortical tumors. J Clin Endocrinol Metab 2012,
97:E100413.
38. Barreau O, Assie G, Wilmot Roussel H, Ragazzon B, Baudry C, Perlemoine K,
et al : Identification of a CpG Island Methylator Phenotype in
Adrenocortical Carcinomas. J Clin Endocrinol Metab 2013, 98(1):E174E184.
39. Gao ZH, Suppola S, Liu J, Heikkila P, Janne J, Voutilainen R: Association of
H19 promoter methylation with the expression of H19 and IGF-II genes
in adrenocortical tumors. J Clin Endocrinol Metab 2002, 87:11706.
40. Byun DS, Lee MG, Chae KS, Ryu BG, Chi SG: Frequent epigenetic
inactivation of RASSF1A by aberrant promoter hypermethylation in
human gastric adenocarcinoma. Cancer Res 2001, 61:70348.
41. Maruyama R, Toyooka S, Toyooka KO, Virmani AK, Zochbauer-Muller S,
Farinas AJ, et al: Aberrant promoter methylation profile of prostate
cancers and its relationship to clinicopathological features. Clin Cancer
Res 2002, 8:5149.
42. Pfeifer GP, Yoon JH, Liu L, Tommasi S, Wilczynski SP, Dammann R:
Methylation of the RASSF1A gene in human cancers. Biol Chem 2002,
383:90714.
43. Yu J, Ni M, Xu J, Zhang HY, Gao BM, Gu JR, et al: M eth yla ti on pr of ilin g o f twe nty
promoter-CpG islands of genes whic h may contribute to hepatocellular
carcinogenesis. Bmc Cancer 2002, 2. doi:10.1186/1471-2407-2-29.
44. van Engeland M, Roemen GM, Brink M, Pachen MM, Weijenberg MP,
de Bruine AP, et al: K-ras mutations and RASSF1A promoter methylation
in colorectal cancer. Oncogene 2002, 21:37925.
45. Yu MY, Tong JHM, Chan PKS, Lee TL, Chan MWY, Chan AWH, et al:
Hypermethylation of the tumor suppressor gene Rassfia and frequent
concomitant loss of heterozygosity at 3p21 in cervical cancers.
Int J Cancer 2003, 105:2049.
46. Astuti D, Agathanggelou A, Honorio S, Dallol A, Martinsson T, Kogner P,
et al : RASSF1A promoter region CpG island hypermethylation in
phaeochromocytomas and neuroblastoma tumours. Oncogene 2001,
20:7573
7.
47. Malpeli G, Amato E, Dandrea M, Fumagalli C, Debattisti V, Boninsegna L,
et al : Methylation-associated down-regulation of RASSF1A and up-
regulation of RASSF1C in pancreatic endocrine tumors. BMC Cancer 2011,
11:351.
48. Gao BN, Xie XJ, Huang CX, Shames DS, Chen TTL, Lewis CM, et al: RASSF1A
polymorphism A133S is associated with early onset breast cancer in
BRCA1/2 mutation carriers. Cancer Res 2008, 68:225.
49. Geli J, Kiss N, Lanner F, Foukakis T, Natalishvili N, Larsson O, et al: The Ras
effectors NORE1A and RASSF1A are frequently inactivated in
pheochromocytoma and abdominal paraganglioma. Endocr Relat Cancer
2007, 14:12534.
50. Geli J, Kiss N, Karimi M, Lee JJ, Backdahl M, Ekstrom TJ, et al: Global and
regional CpG methylation in pheochromocytomas and abdominal
paragangliomas: association to malignant behavior. Clin Cancer Res 2008,
14:25519.
51. Rong R, Jin W, Zhang J, Sheikh MS, Huang Y: Tumor suppressor RASSF1A
is a microtubule-binding protein that stabilizes microtubules and
induces G2/M arrest. Oncogene 2004, 23:821630.
52. Vos MD, Martinez A, Elam C, Dallol A, Taylor BJ, Latif F, et al: A role for the
RASSF1A tumor suppressor in the regulation of tubulin polymerization
and genomic stability. Cancer Res 2004, 64:424450.
53. El-Kalla M, Onyskiw C, Baksh S: Functional importance of RASSF1A
microtubule localization and polymorphisms. Oncogene 2010, 29:572940.
54. Agathanggelou A, Honorio S, Macartney DP, Martinez A, Dallol A, Rader J,
et al : Methylation associated inactivation of RASSF1A from region 3p21.3
in lung, breast and ovarian tumours. Oncogene 2001, 20:150918.
55. Korah R, Choi L, Barrios J, Wieder R: Expression of FGF-2 alters focal
adhesion dynamics in migration-restricted MDA-MB-231 breast cancer
cells. Breast Cancer Res Treat 2004, 88(1):1728.
doi:10.1186/1476-4598-12-87
Cite this article as: Korah et al.: Epigenetic silencing of RASSF1A
deregulates cytoskeleton and promotes malignant behavior of
adrenocortical carcinoma. Molecular Cancer 2013 12:87.
Submit your next manuscript to BioMed Central
and take full advantage of:
Convenient online submission
Thorough peer review
No space constraints or color figure charges
Immediate publication on acceptance
Inclusion in PubMed, CAS, Scopus and Google Scholar
Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Korah et al. Molecular Cancer 2013, 12:87 Page 13 of 13
http://www.molecular-cancer.com/content/12/1/87
    • "One of the possible mechanisms of microtubule stabilization by RASSF1A involves RAN GTPase [22] , and another mechanism discovered recently includes the suppression of histone deacetylase 6 which functions as a tubulin deacetylase [23]. The ability of RASSF1A to regulate microtubule stability, spindle assembly and chromosome attachment determine RASSF1A tumor suppressor functions towards controlling cell growth, transformation , motility and invasiveness [15,19,24]. Further studies have shown that interaction of RASSF1A with RABP1 (RASSF1A binding protein 1/C19ORF5/MAP1S) leads to its recruitment to the spindle poles in pro-metaphase and its interaction with Cdc20 [25] (please note that here is a contradictory report on RASSF1A interaction with Cdc20 [26]), inhibition of APC (anaphase promoting complex), accumulation of mitotic cyclins A and B and eventually mitotic arrest [25,27,28] . "
    [Show abstract] [Hide abstract] ABSTRACT: Genetic changes through allelic loss and nucleic acid or protein modifications are the main contributors to loss of function of tumor suppressor proteins. In particular, epigenetic silencing of genes by promoter hypermethylation is associated with increased tumor severity and poor survival. The RASSF (Ras association domain family) family of proteins consists of 10 members, many of which are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers such as leukemia, melanoma, breast, prostate, neck, lung, brain, colorectal and kidney cancers. In addition to their tumor suppressor function, RASSF proteins act as scaffolding agents in microtubule stability, regulate mitotic cell division, modulate apoptosis, control cell migration and cell adhesion, and modulate NFκB activity and the duration of inflammation. The ubiquitous functions of these proteins highlight their importance in numerous physiological pathways. In this review, we will focus on the biological roles of the RASSF family members and their regulation.
    Full-text · Article · Mar 2014
    • "One of the possible mechanisms of microtubule stabilization by RASSF1A involves RAN GTPase [22], and another mechanism discovered recently includes the suppression of histone deacetylase 6 which functions as a tubulin deacetylase [23]. The ability of RASSF1A to regulate microtubule stability, spindle assembly and chromosome attachment determine RASSF1A tumor suppressor functions towards controlling cell growth, transformation , motility and invasiveness [15] [19] [24]. "
    [Show abstract] [Hide abstract] ABSTRACT: The Ras-association domain family (RASSF) proteins are tumor suppressor proteins whose importance to the development of cancer has become increasingly apparent over the last 12 years. While possessing no enzymatic activity, they appear to function as scaffolding molecules to regulate the activity of a surprisingly broad array of effectors. They are implicated in the regulation of a diverse range of biological functions including apoptosis, autophagy, cell cycle control, microtubule dynamics, and DNA repair. In addition, they are thought to be one of the regulators of the Hippo pathway, the newly emerging tumor suppressor pathway evolutionarily conserved between Drosophila and mammals.
    Full-text · Article · Dec 2012
  • [Show abstract] [Hide abstract] ABSTRACT: A reappraisal of the major advances in the diagnostic pathology of adrenal cortical lesions and tumors in the last 25 years is presented, with special reference to the definition of malignancy in primary adrenal cancer and its variants. Slightly more than 25 years ago, Weiss proposed his diagnostic scoring system for adrenal cortical carcinoma. This represented a milestone for adrenal pathologists and the starting point for further modifications of the system, either through minor changes in the scoring procedure itself or concentrating on some particular Weiss criterion such as mitotic index, integrated into alternative scoring schemes or algorithms that are currently under validation. Improvements in diagnostic immunohistochemistry have led to the identification of markers of cortical origin, such as Melan-A, alpha-inhibin, and SF-1 and of prognostic factors in carcinoma, such as the Ki-67 proliferation index and SF-1 itself. With regard to hyperplastic conditions, genetic investigations have allowed the association of the majority of cases of primary pigmented nodular adrenocortical disease (PPNAD) in Carney complex to mutations in the gene encoding the regulatory subunit 1A of protein kinase A (PRKAR1A). Other hereditary conditions are also associated with adrenal cortical tumors, including the Li-Fraumeni, Beckwith-Wiedemann, Gardner, multiple endocrine neoplasia type 1, and neurofibromatosis type 1 syndromes. Moreover, several advances have been made in the knowledge of the molecular background of sporadic tumors, and a number of molecules/genes are of particular interest as potential diagnostic and prognostic biomarkers.
    Article · Jan 2014
Show more