Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm
Identifieur interne : 004705 ( Pmc/Corpus ); précédent : 004704; suivant : 004706Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in the cytoplasm
Auteurs : Diana Golden ; Lloyd G. CantleySource :
- Oncogene [ 0950-9232 ] ; 2014.
Abstract
Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during
developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory
has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it
promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2
phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and
phosphorylates this site
Url:
DOI: 10.1038/onc.2014.395
PubMed: 25486430
PubMed Central: 4459945
Links to Exploration step
PMC:4459945Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in
the cytoplasm</title>
<author><name sortKey="Golden, Diana" sort="Golden, Diana" uniqKey="Golden D" first="Diana" last="Golden">Diana Golden</name>
<affiliation><nlm:aff id="A1">Center for Vascular Biology, University of Connecticut Health Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Cantley, Lloyd G" sort="Cantley, Lloyd G" uniqKey="Cantley L" first="Lloyd G." last="Cantley">Lloyd G. Cantley</name>
<affiliation><nlm:aff id="A2">Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine</nlm:aff>
</affiliation>
</author>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in
the cytoplasm</title>
<author><name sortKey="Golden, Diana" sort="Golden, Diana" uniqKey="Golden D" first="Diana" last="Golden">Diana Golden</name>
<affiliation><nlm:aff id="A1">Center for Vascular Biology, University of Connecticut Health Center</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Cantley, Lloyd G" sort="Cantley, Lloyd G" uniqKey="Cantley L" first="Lloyd G." last="Cantley">Lloyd G. Cantley</name>
<affiliation><nlm:aff id="A2">Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Oncogene</title>
<idno type="ISSN">0950-9232</idno>
<idno type="eISSN">1476-5594</idno>
<imprint><date when="2014">2014</date>
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<front><div type="abstract" xml:lang="en"><p id="P1">Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during
developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory
has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it
promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2
phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and
phosphorylates this site <italic>in vitro</italic>
. Knock-down or inhibition of the
CK2α/α′ kinase subunit in epithelial cells causes de novo
accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes
constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an
S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial
maintenance. In malignant breast cancer cells the CK2β regulatory subunit is
downregulated and FOXC2 is found in the nucleus, correlating with an increase in
α-SMA expression. Restoration of CK2β expression in these cells results in
cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell
migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial
cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased
α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on
these findings we propose that Foxc2 is functionally maintained in the cytoplasm of normal
epithelial cells by CK2α/α′-mediated phosphorylation at serine 124
that is dependent on proper targeting of the holoenzyme via the CK2β regulatory
subunit.</p>
</div>
</front>
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</back>
</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">8711562</journal-id>
<journal-id journal-id-type="pubmed-jr-id">6325</journal-id>
<journal-id journal-id-type="nlm-ta">Oncogene</journal-id>
<journal-id journal-id-type="iso-abbrev">Oncogene</journal-id>
<journal-title-group><journal-title>Oncogene</journal-title>
</journal-title-group>
<issn pub-type="ppub">0950-9232</issn>
<issn pub-type="epub">1476-5594</issn>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">25486430</article-id>
<article-id pub-id-type="pmc">4459945</article-id>
<article-id pub-id-type="doi">10.1038/onc.2014.395</article-id>
<article-id pub-id-type="manuscript">NIHMS627033</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Casein Kinase 2 prevents mesenchymal transformation by maintaining Foxc2 in
the cytoplasm</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Golden</surname>
<given-names>Diana</given-names>
</name>
<degrees>PhD</degrees>
<xref ref-type="aff" rid="A1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Cantley</surname>
<given-names>Lloyd G.</given-names>
</name>
<degrees>MD</degrees>
<xref ref-type="aff" rid="A2">2</xref>
</contrib>
</contrib-group>
<aff id="A1"><label>1</label>
Center for Vascular Biology, University of Connecticut Health Center</aff>
<aff id="A2"><label>2</label>
Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine</aff>
<author-notes><corresp id="FN1">Correspondence: Diana Golden, PhD, University of Connecticut Health
Center, Center for Vascular Biology, 263 Farmington Ave, Farmington, CT 06030-3501,
(860)-604-6144, <email>dgolden@uchc.edu</email>
</corresp>
</author-notes>
<pub-date pub-type="nihms-submitted"><day>11</day>
<month>9</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="epub"><day>08</day>
<month>12</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="ppub"><day>3</day>
<month>9</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>03</day>
<month>3</month>
<year>2016</year>
</pub-date>
<volume>34</volume>
<issue>36</issue>
<fpage>4702</fpage>
<lpage>4712</lpage>
<pmc-comment>elocation-id from pubmed: 10.1038/onc.2014.395</pmc-comment>
<permissions><license xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms"><license-p>Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:<ext-link ext-link-type="uri" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</ext-link>
</license-p>
</license>
</permissions>
<abstract><p id="P1">Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during
developmental EMT and has been associated with EMT in malignant epithelia. Our laboratory
has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it
promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2
phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and
phosphorylates this site <italic>in vitro</italic>
. Knock-down or inhibition of the
CK2α/α′ kinase subunit in epithelial cells causes de novo
accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes
constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an
S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial
maintenance. In malignant breast cancer cells the CK2β regulatory subunit is
downregulated and FOXC2 is found in the nucleus, correlating with an increase in
α-SMA expression. Restoration of CK2β expression in these cells results in
cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell
migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial
cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased
α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on
these findings we propose that Foxc2 is functionally maintained in the cytoplasm of normal
epithelial cells by CK2α/α′-mediated phosphorylation at serine 124
that is dependent on proper targeting of the holoenzyme via the CK2β regulatory
subunit.</p>
</abstract>
<kwd-group><kwd>Foxc2</kwd>
<kwd>E-cadherin</kwd>
<kwd>Epithelial Cell</kwd>
<kwd>Epithelial to Mesenchymal Transition</kwd>
<kwd>CK2 Signaling</kwd>
</kwd-group>
</article-meta>
</front>
<body><sec sec-type="intro" id="S1"><title>INTRODUCTION</title>
<p id="P2">Cells in a mature epithelium such as the kidney tubule or breast acini are
maintained in a highly differentiated state. However, following organ injury these cells
undergo transient de-differentiation during which they lose their brush border, exhibit a
flattened morphology and express mesenchymal markers such as vimentin and α-smooth
muscle actin.<sup><xref rid="R1" ref-type="bibr">1</xref>
–<xref rid="R3" ref-type="bibr">3</xref>
</sup>
This de-differentiation is believed to promote cell
survival, migration and proliferation, thus enhancing regeneration of the mature
epithelium.<sup><xref rid="R4" ref-type="bibr">4</xref>
</sup>
However, sustained
de-differentiation (often referred to as epithelial–mesenchymal transition or EMT)
can lead to fibrosis and scarring following organ injury<sup><xref rid="R5" ref-type="bibr">5</xref>
–<xref rid="R6" ref-type="bibr">6</xref>
</sup>
and can promote tumor
metastasis in epithelial cancers.<sup><xref rid="R14" ref-type="bibr">14</xref>
</sup>
Identifying the factors that regulate the processes of dedifferentiation and
redifferentiation is therefore crucial to understanding normal epithelial biology and the
forces that underlie aberrant tumor behavior.</p>
<p id="P3">Fox (forkhead box) proteins are a family of transcription factors that are
important in regulating the expression of genes involved in cell growth, proliferation,
differentiation and longevity.<sup><xref rid="R7" ref-type="bibr">7</xref>
–<xref rid="R9" ref-type="bibr">9</xref>
</sup>
Fox proteins contain the FHD, a sequence of ~100
amino acids forming a motif that binds to DNA.<sup><xref rid="R10" ref-type="bibr">10</xref>
</sup>
Foxc2, belonging to the ‘C’ subfamily, is required for
cardiovascular development<sup><xref rid="R11" ref-type="bibr">11</xref>
</sup>
, early
organogenesis of the kidney<sup><xref rid="R12" ref-type="bibr">12</xref>
</sup>
, podocyte
differentiation and glomerular basement membrane maturation.<sup><xref rid="R13" ref-type="bibr">13</xref>
</sup>
Mani et al.<sup><xref rid="R14" ref-type="bibr">14</xref>
</sup>
have demonstrated that Foxc2 can act as an activator of epithelial cell
dedifferentiation during tumor metastasis, while Bard and colleagues<sup><xref rid="R15" ref-type="bibr">15</xref>
</sup>
reported a possible function for Foxc2 in epithelial
cell differentiation. Our laboratory previously investigated Foxc2 expression and
localization following ischemia/reperfusion injury (I/R) of the kidney.<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
Foxc2 was transiently upregulated in tubular epithelial
cells after ischemic injury but was detectable only in the cytoplasm of these cells both
<italic>in vitro</italic>
and <italic>in vivo</italic>
. Suppression of cytoplasmic Foxc2
using RNAi resulted in a partial loss of the epithelial phenotype. In contrast, ex vivo
over-expression of Foxc2 in epithelial cells resulted in nuclear translocation and promotion
of a mesenchymal phenotype. These results suggest that the mechanism by which epithelial
cells control Foxc2 localization is critical for maintaining the epithelial phenotype.</p>
<p id="P4">The amino terminus of Foxc2 contains a region within the FHD that is known to be
mutated in patients with autosomal dominant inherited primary lymphedema associated with
distichiasis.<sup><xref rid="R17" ref-type="bibr">17</xref>
–<xref rid="R19" ref-type="bibr">19</xref>
</sup>
One of those mutations, R120H, has been found to alter
the subcellular localization of Foxc2 by an as yet undetermined mechanism. Our analysis of
that region of Foxc2 reveals that it is adjacent to a potential casein kinase 2
phosphorylation site at serine 124. In the current study we show that CK2 can phosphorylate
Foxc2 in vitro and that knock-down or inhibition of CK2 in cultured epithelial cells causes
translocation of endogenous Foxc2 to the nucleus. Foxc2-S124D, containing a phosphomimetic
of the putative CK2 phosphorylation site, was constitutively expressed in the cytoplasm
whereas Foxc2-S124L, a non-phosphorylatable mutant, was found predominantly in the nucleus
and induced mesenchymal gene expression. In highly malignant breast cancer cells FOXC2 was
found in the nucleus, correlating with increased expression of mesenchymal markers.
Examination of CK2 expression in these cells revealed down-regulation of the CK2 regulatory
subunit, CK2β. Over-expression of CK2β induced cytoplasmic localization of
FOXC2, suppressed α-SMA expression, reduced cellular migration and invasion, whereas
knockdown of CK2β in normal breast epithelial cells led to nuclear localization of
FOXC2, downregulation of E-Cadherin, upregulation of mesenchymal markers, and increased cell
migration and invasion. The ability of over-expressed CK2β to suppress the
mesenchymal phenotype of malignant breast epithelial cells was prevented by the
co-expression of Foxc2-S124L, confirming that Foxc2 is a key target of CK2β in
epithelial maintenance.</p>
</sec>
<sec sec-type="results" id="S2"><title>RESULTS</title>
<sec id="S3"><title>Foxc2 localization</title>
<p id="P5">Cell fractionation of cultured MPT cells (immortalized epithelial cells derived
from the mouse proximal tubule)<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
revealed that Foxc2 was only detectable in the cytoplasm, with no protein seen in the
nuclear fraction (<xref rid="F1" ref-type="fig">Figure 1A</xref>
). In contrast, Foxc2 was
localized in both the cytoplasm and nucleus of NIH3T3 fibroblasts. These results are
consistent with our immunofluorescent analysis of renal tubular epithelial cells in
vivo<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
and suggest that epithelial
cells normally maintain Foxc2 in the cytoplasm. Transfection of MPT cells with a vector
encoding FLAG-tagged Foxc2 resulted in over-expression of Foxc2 with de novo detection of
Foxc2 in the nucleus of MPT cells (<xref rid="F1" ref-type="fig">Figure 1A</xref>
).
Examination of the sequence of Foxc2 revealed that the serine at position 124
(<sup>124</sup>
SLNE) is a potential phosphorylation site for casein kinase 2 (CK2,
consensus sequence pS/TXXE/D). To determine whether phosphorylation at this site might
regulate Foxc2 localization, constructs encoding full length GFP-Foxc2-S124L (preventing
phosphorylation of the site) and GFP-Foxc2-S124D (mimicking constitutive phosphorylation)
were generated. Transient transfection of MPT cells with these constructs again
demonstrated that over-expressed wild-type GFP-Foxc2 localized to both the nuclear and
cytoplasmic compartments, whereas GFP-Foxc2-S124L localized predominantly to the nucleus
in a speckled pattern and GFP-Foxc2-S124D localized to the cytoplasm (<xref rid="F1" ref-type="fig">Figure 1B, C</xref>
). Side-by-side comparison of whole cell lysates
confirmed that all three constructs were expressed at similar levels (<xref rid="SD2" ref-type="supplementary-material">Supplemental Figure 1</xref>
). These results suggest
that phosphorylation at serine 124 promotes cytoplasmic localization of Foxc2.</p>
</sec>
<sec id="S4"><title>CK2 is required to maintain cytoplasmic localization of endogenous Foxc2</title>
<p id="P6">To determine whether CK2 might be responsible for phosphorylation at S124, we
first examined the potential association of CK2 with Foxc2. GFP-Foxc2 was transfected into
MPT cells and the fusion protein immunoprecipitated with anti-GFP. These experiments
revealed that GFP-Foxc2 co-immunoprecipitates with endogenous CK2 in renal epithelial
cells (<xref rid="F2" ref-type="fig">Figure 2A</xref>
). MPT cells were then transfected
with either siRNA directed against the CK2 kinase subunits
(CK2α/α′) or scrambled siRNA (<xref rid="F2" ref-type="fig">Figure
2B</xref>
, 65% knock-down of CK2α/α′), followed by
analysis of the subcellular localization of endogenous Foxc2 24 hours later. Cell
fractionation confirmed that endogenous Foxc2 is detectable only in the cytoplasm of
control MPT cells, whereas CK2α/α′ knock-down resulted in
decreased cytoplasmic localization and de novo detection of Foxc2 in the nucleus (<xref rid="F2" ref-type="fig">Figure 2C</xref>
, quantified below). To determine whether the
kinase activity of CK2 is required for cytosolic maintenance of Foxc2, MPT cells were
treated ± TBCA (a cell-permeable CK2 kinase inhibitor) <sup><xref rid="R20" ref-type="bibr">20</xref>
</sup>
or vehicle followed by examination of the localization
of endogenous Foxc2. Cell fractionation revealed that Foxc2 was found in the cytoplasm of
control MPT cells, whereas treatment with TBCA for 1 hour resulted in decreased
cytoplasmic localization and obvious detection of Foxc2 in the nucleus (<xref rid="F2" ref-type="fig">Figure 2D</xref>
). These results demonstrate that CK2 associates with
Foxc2 and maintains its cytoplasmic localization in a kinase dependent manner.</p>
</sec>
<sec id="S5"><title>CK2 dependent cytoplasmic localization of Foxc2 requires serine 124</title>
<p id="P7">To determine whether CK2-mediated localization of Foxc2 is dependent on serine
124, MPT cells were transiently transfected with either full-length GFP-Foxc2 or
GFP-Foxc2-S124D ± the CK2 kinase inhibitor TBCA. As described above, in the
absence of TBCA over-expressed GFP-Foxc2 localized to both the cytoplasm and nucleus
whereas GFP-Foxc2-S124D localized to the cytoplasm (<xref rid="F2" ref-type="fig">Figure
2E</xref>
). Following CK2 inhibition with TBCA, GFP-Foxc2 was found almost entirely in
the nucleus whereas GFP-Foxc2-S124D remained localized in the cytoplasm (<xref rid="F2" ref-type="fig">Figure 2F</xref>
, quantified below). These results demonstrate that, in
contrast to endogenous Foxc2, the S124D phosphomimetic does not require CK2 kinase
activity for cytoplasmic localization, suggesting that CK2 may normally phosphorylate this
site.</p>
<p id="P8">To determine if Foxc2 is indeed a CK2 substrate, an <italic>in vitro</italic>
kinase assay was performed in which either immunoprecipitated GFP-Foxc2 or GFP-Foxc2-S124L
was used as a substrate for purified CK2α/α′/β holoenzyme.
GFP-Foxc2 was phosphorylated in the presence of added CK2, with less phosphorylation of
GFP-Foxc2-S124L (<xref rid="F3" ref-type="fig">Figure 3</xref>
). These results show that
CK2 can phosphorylate Foxc2 and suggest that serine 124 is the primary CK2 phosphorylation
residue.</p>
</sec>
<sec id="S6"><title>Cytoplasmic localization of Foxc2 maintains the epithelial phenotype</title>
<p id="P9">In normal renal epithelia, Foxc2 is upregulated after kidney injury, but remains
localized to the cytoplasm.<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
In
contrast, over-expression of Foxc2 by transfection or in epithelial tumors results in
increased nuclear localization and a mesenchymal expression pattern (<xref rid="F1" ref-type="fig">Figure 1A</xref>
).<sup><xref rid="R14" ref-type="bibr">14</xref>
, <xref rid="R16" ref-type="bibr">16</xref>
</sup>
To determine whether nuclear translocation
of Foxc2 is required for mesenchymal expression, MPT cells were transfected with either
GFP alone, GFP-Foxc2-S124D or GFP-Foxc2-S124L, followed by examination of cell morphology
and expression of epithelial and mesenchymal markers. Transfection with GFP-Foxc2-S124L
(found predominantly in the nucleus) resulted in downregulation of E-cadherin and
upregulation of both vimentin and α-SMA as compared to control cells transfected
with GFP alone (<xref rid="F4" ref-type="fig">Figure 4A</xref>
, quantified in B). In
contrast, cells over-expressing GFP-Foxc2-S124D (found predominantly in the cytoplasm)
exhibited a modest increase in E-cadherin expression and failed to upregulate either
vimentin or α-SMA, even though the total amount of GFP-Foxc2 was indistinguishable
in the two groups. F-actin staining (<xref rid="SD3" ref-type="supplementary-material">Supplemental Figure 2</xref>
) revealed an epithelial morphology in MPT cells
transfected with GFP only or GFP-Foxc2-S124D, while more of a spindled shape was observed
in the GFP-Foxc2 and GFP-Foxc2-S124L expressing cells.</p>
<p id="P10">To determine if the difference in protein expression observed between
Foxc2-S124D and Foxc2-S124L transfected cells was due to transcriptional regulation,
qRT-PCR was performed to quantify mRNA. These studies demonstrated that cells expressing
Foxc2-S124L exhibited a significant reduction in the mRNA for E-cadherin and increased
mRNA for both vimentin and α-SMA as compared to either control cells or those
expressing GFP-Foxc2-S124D (<xref rid="F4" ref-type="fig">Figure 4C</xref>
). Cumulatively
these data suggest that activation of a mesenchymal expression phenotype by Foxc2 requires
trafficking to the nucleus and subsequent regulation of gene transcription.</p>
</sec>
<sec id="S7"><title>FOXC2 nuclear localization is increased in metastatic breast cancer cells</title>
<p id="P11">Increased FOXC2 expression in breast cancer biopsies has been shown to correlate
with increased metastasis.<sup><xref rid="R14" ref-type="bibr">14</xref>
</sup>
To
determine whether FOXC2 is localized differently in normal breast epithelial cells as
compared to cells derived from metastatic tumors, we compared MCF10A cells derived from
normal human breast tissue with MDA-MB-436 (derived from a weakly metastatic tumor) and
MDA-MB-231 (derived from a highly metastatic tumor) breast cancer cell lines.<sup><xref rid="R29" ref-type="bibr">29</xref>
</sup>
Whole cell lysates demonstrated an increase
in total FOXC2 expression in highly metastatic MDA-MB-231 cells as compared to the normal
breast MCF10A cells and weakly metastatic MDA-MB-436 cells (<xref rid="F5" ref-type="fig">Figure 5A</xref>
, quantified below). Subcellular fractionation reveals that FOXC2 is
detected only in the cytoplasm of MCF10A cells, similar to the pattern seen in normal
renal tubular epithelial cells (<xref rid="F5" ref-type="fig">Figure 5B</xref>
, quantified
below). In contrast, MDA-MB-436 cells had mixed cytosolic and nuclear expression, while
MDA-MB-231 cells expressed FOXC2 predominantly in the nuclear compartment. Thus FOXC2 is
easily detected in the nucleus in cells derived from metastatic tumors, but is maintained
in the cytoplasm of normal breast epithelial cells.</p>
</sec>
<sec id="S8"><title>Metastatic breast cancer cell lines have decreased CK2β and upregulate
mesenchymal proteins</title>
<p id="P12">Over-expression of transfected Foxc2 in normal renal epithelia leads to nuclear
localization, <sup><xref rid="R14" ref-type="bibr">14</xref>
, <xref rid="R16" ref-type="bibr">16</xref>
</sup>
and high levels of endogenous Foxc2 in MDA-MB-231
cells correlates with nuclear localization (<xref rid="F5" ref-type="fig">Figure
5</xref>
). Surprisingly, MDA-MB-436 cells also exhibited nuclear localization of Foxc2
despite having normal levels of the protein (<xref rid="F5" ref-type="fig">Figure
5A</xref>
). Recently, Deshiere et al.<sup><xref rid="R28" ref-type="bibr">28</xref>
</sup>
showed that unbalanced expression of CK2α and β
subunits in a subset of breast tumor samples correlated with expression of EMT-related
markers. We therefore examined CK2 subunit expression and mesenchymal markers in the
metastatic MDA-MB-436 and MDA-MB-231 cells as compared to non-malignant MCF10A breast
epithelial cells and MPT kidney epithelial cells. Similar to MPT cells, MCF10A cells
expressed high levels of E-cadherin and low levels of the mesenchymal proteins vimentin
and α-SMA (<xref rid="F6" ref-type="fig">Figure 6A</xref>
, quantified in B).
Consistent with the findings of Deshiere et al., <sup><xref rid="R28" ref-type="bibr">28</xref>
</sup>
these cells had the highest expression of CK2β with a CK2
α/α′ to β ratio of 2:5. In contrast, both MDA-MB-436 and
231 cells exhibited downregulation of CK2β and upregulation of
α/α′, resulting in a reversal of the α/α′
to β ratio to 3:1 and 10:1, respectively. This correlated with suppression of
E-cadherin expression and upregulation of vimentin and α-SMA in the metastatic
cell lines. This same reversal of the α/α′:β ratio was
detected when MDA-MB-231 cells were compared to non-malignant kidney MPT cells (<xref rid="F6" ref-type="fig">Figure 6C</xref>
).</p>
<p id="P13">NIH3T3 fibroblasts, which express Foxc2 in the nucleus and exhibit a mesenchymal
phenotype, were also analyzed for their Foxc2, CK2 α/α′ and
CK2β expression. Similar to MDA-MB-436 cells, NIH3T3 cells had total Foxc2
expression levels comparable to those found in non-malignant MPT and MCF10A epithelial
cells, but exhibited increased CK2α/α′ expression with low levels
of CK2β expression (<xref rid="F6" ref-type="fig">Figure 6C</xref>
). These data
suggest that downregulation of CK2β may be sufficient to prevent CK2-mediated
maintenance of Foxc2 in the cytoplasm, even in the setting of normal levels of total Foxc2
expression.</p>
</sec>
<sec id="S9"><title>Over-expressing CK2β in malignant breast cancer cells rescues FOXC2
cytoplasmic localization</title>
<p id="P14">In light of the correlation between downregulation of CK2β and nuclear
localization of FOXC2, we pursued the possibility that normalization of CK2β
expression in MDA-MB-231 cells could promote FOXC2 localization to the cytoplasm.
MDA-MB-231 cells transfected with a vector encoding CK2β demonstrated an increase
in CK2β expression as well as a decrease in CK2α/α′ (<xref rid="F7" ref-type="fig">Figure 7A</xref>
). This partial normalization of the
CK2α/α′:CK2β ratio led to a decrease in α-SMA
expression by the MDA-MB-231 cells, but failed to reduce their vimentin expression or
rescue E-cadherin expression. Subcellular fractionation revealed that re-expression of
CK2β in MDA-MB-231 cells induced a shift of most of the FOXC2 from the nucleus to
the cytoplasm, although FOXC2 was still detected in the nucleus at low levels (<xref rid="F7" ref-type="fig">Figure 7B</xref>
). This resulted in a decrease in both serum
dependent and serum-independent cell migration (<xref rid="F7" ref-type="fig">Figure
7C</xref>
) and cell invasion through a basement membrane extract (<xref rid="F7" ref-type="fig">Figure 7D</xref>
). These data suggest that reduced levels of CK2β
may impair CK2α/α′-mediated FOXC2 phosphorylation, thus promoting
FOXC2 nuclear localization and an increase in cellular migration and invasion.</p>
</sec>
<sec id="S10"><title>Reducing CK2β expression in normal epithelial cells promotes FOXC2 nuclear
localization and a mesenchymal phenotype</title>
<p id="P15">To better clarify the role of CK2β in regulating CK2-dependent FOXC2
localization, MCF10A cells were transfected with siRNA against CK2β. This resulted
in decreased CK2β expression and an increase in CK2α/α′
(<xref rid="F8" ref-type="fig">Figure 8A</xref>
), similar to the pattern seen in
MDA-MB-436 and MDA-MB-231 cells (<xref rid="F6" ref-type="fig">Figure 6A</xref>
). This
reversal of the CK2α/α′:CK2β ratio resulted in
translocation of FOXC2 from the cytoplasm to the nucleus (<xref rid="F8" ref-type="fig">Figure 8B</xref>
), along with downregulation of E-cadherin and upregulation of
α-SMA (<xref rid="F8" ref-type="fig">Figure 8A</xref>
). Functionally, these
CK2β knock-down cells exhibited increased cell migration and increased cell
invasion (<xref rid="F8" ref-type="fig">Figure 8C, D</xref>
). These data demonstrate that
maintenance of the proper ratio of CK2β to CK2α/α′ in
epithelial cells is required for maintenance of FOXC2 in the cytoplasm and prevention of
EMT.</p>
</sec>
<sec id="S11"><title>FOXC2 is a critical CK2 target for epithelial maintenance</title>
<p id="P16">To determine the functional significance of FOXC2 phosphorylation in the overall
spectrum of CK2 signaling, MCF10A cells were transfected with either WT Foxc2 or
Foxc2-S124L ± CK2β. As predicted from the results in kidney epithelial
cells, over-expression of either Foxc2 or Foxc2-S124L alone led to a marked increase in
MCF10A cell migration and invasion (<xref rid="F9" ref-type="fig">Figure 9A, C</xref>
).
This increase in mesenchymal behavior was suppressed in cells co-expressing CK2β
along with wild-type Foxc2, whereas CK2β failed to suppress the increased
migration and matrix invasion of Foxc2-S124L expressing MCF10A cells.</p>
<p id="P17">In the metastatic MDA-MB-231 breast cancer cell line (in which Foxc2 is already
localized to the nucleus), migratory and matrix invasion rates were high at baseline and
did not increase further with expression of Foxc2 or Foxc2-S124L alone (<xref rid="F9" ref-type="fig">Figure 9B, D</xref>
). As before, over-expression of CK2β
suppressed the migratory and invasive phenotype of these cells, and this suppressive
effect was maintained in cells in which CK2β was co-expressed with wild-type
Foxc2. In contrast, over-expression of CK2β in cells co-expressing Foxc2-S124L
failed to suppress migration and invasion (<xref rid="F9" ref-type="fig">Figure 9B,
D</xref>
). These data demonstrate that cytoplasmic maintenance of Foxc2 plays a key
effector role in CK2-dependent epithelial maintenance.</p>
</sec>
</sec>
<sec sec-type="discussion" id="S12"><title>DISCUSSION</title>
<p id="P18">Our previous results revealed that normal epithelial cells maintain Foxc2 in the
cytoplasm even when Foxc2 is physiologically upregulated following epithelial
injury.<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
However, when Foxc2 is
over-expressed, such as following transfection of cultured cells or in epithelial-derived
tumors, it can be readily detected in both the cytoplasm and the nucleus.<sup><xref rid="R14" ref-type="bibr">14</xref>
, <xref rid="R16" ref-type="bibr">16</xref>
</sup>
These results suggest that Foxc2 is maintained in the cytoplasm of normal epithelial cells
by a regulated signaling pathway that can either be overwhelmed in the setting of very high
levels of Foxc2 and/or dysregulated following transition to a malignant phenotype. In the
current study we demonstrate that a core feature of this signaling pathway is the
CK2α/α′-dependent phosphorylation of Foxc2 at serine 124 in the
amino terminus of the protein, and that this process is regulated in a cell-type specific
manner by the relative expression of the CK2α/α′ and CK2β
subunits.</p>
<p id="P19">Serine 124 sits in a highly conserved region of Foxc2 that is also present in
other Fox family members (<xref rid="T1" ref-type="table">Table 1</xref>
). Analysis of the
flanking sequence around serine 124 reveals that this is a potential CK2 phosphorylation
site. Our results show that CK2 associates with Foxc2, and that in the absence of CK2 or
following inhibition of CK2 kinase activity, Foxc2 predominantly traffics into the nucleus
even in the absence of over-expression. These observations provide strong evidence that
Foxc2 is maintained in the cytoplasm in a CK2-dependent manner, but do not define the CK2
regulatory site. To address this, we generated a non-phosphorylatable mutant of Foxc2 that
mimics one of the mutations seen in patients with LD (S124L) as well as a phosphomimetic
mutation at the site (S124D) and compared the localization of these constructs with that of
wild-type Foxc2. Consistent with the model that phosphorylation of Foxc2 at serine 124
specifically maintains Foxc2 in the cytoplasm, S124D mutants were almost exclusively
localized to the cytoplasm whereas S124L mutants were predominantly found in the nucleus.
The additional observation that treatment of cells expressing the S124D mutant with the CK2
inhibitor TBCA did not cause Foxc2-S124D to traffic into the nucleus suggests that serine
124 is the CK2-dependent phosphorylation site that specifically mediates cytoplasmic
retention of Foxc2. This model is supported by our <italic>in vitro</italic>
kinase assay
demonstrating that CK2 directly phosphorylates Foxc2, with reduced phosphorylation of the
S124L mutant.</p>
<p id="P20">CK2 is a constitutively active kinase that participates in many cellular processes
including replication, transcription, translation and signal transduction.<sup><xref rid="R21" ref-type="bibr">21</xref>
</sup>
CK2 consists of a heterodimer of the
CK2α serine/threonine kinase and the CK2β regulatory subunit that is
required for substrate binding. <sup><xref rid="R21" ref-type="bibr">21</xref>
</sup>
CK2 has
been described as a nuclear protein kinase but has been found to be present in both the
cytosol and the nucleus.<sup><xref rid="R22" ref-type="bibr">22</xref>
</sup>
Consistent with
a functional role of CK2 in both locations, Cdc25 has been shown to be exported from the
nucleus by CK2 dependent phosphorylation, <sup><xref rid="R23" ref-type="bibr">23</xref>
</sup>
while CK2-mediated phosphorylation of survivin and Pparγ has been
implicated in maintenance of their cytoplasmic localization.<sup><xref rid="R24" ref-type="bibr">24</xref>
–<xref rid="R26" ref-type="bibr">26</xref>
</sup>
</p>
<p id="P21">The mechanism by which CK2-dependent phosphorylation of Foxc2 is regulated appears
to be similar to that described for CK2-dependent phosphorylation of Snail. MacPherson et
al.<sup><xref rid="R27" ref-type="bibr">27</xref>
</sup>
demonstrated that CK2
phosphorylates Snail1 to regulate its transcriptional activity and Deshiere and colleagues
<sup><xref rid="R28" ref-type="bibr">28</xref>
</sup>
found that the level of expression
of the CK2β regulatory subunit was critical in determining the extent of Snail1
phosphorylation. CK2β regulates binding of CK2α/α′ to target
proteins, and Deshiere et al.<sup><xref rid="R28" ref-type="bibr">28</xref>
</sup>
found that
low levels of CK2β resulted in failure of CK2α/α′ dependent
phosphorylation of Snail and subsequent Snail stabilization and induction of EMT. Our
results are consistent with these findings in that downregulation of CK2β was
detected in both of the breast metastatic tumor cell lines that we examined, resulting in a
progressive reversal of the CK2α/α′ to β subunit ratio (as
determined using our antibodies) in moving from normal breast epithelia (2:5) to weakly
metastatic (3:1) and highly metastatic (10:1) breast tumor cells. This correlated with
increased nuclear localization of FOXC2 even in the absence of FOXC2 over-expression.
Mesenchymal fibroblasts, which express Foxc2 in both compartments, also demonstrated a
decrease in β subunit expression relative to α.</p>
<p id="P22">More interestingly, re-expression of the CK2β subunit in MDA-MB-231 cells
partially normalized the α/α′:β subunit ratio, induced
cytoplasmic localization of the majority of FOXC2, and reduced migration and invasion of
these highly metastatic breast cancer cells. However, co-expressing the non-phosphorylatable
Foxc2-S124L mutant along with CK2β significantly suppressed the ability of CK2 to
restore a more epithelial phenotype in MDA-MB-231 cells and promoted a mensenchymal
phenotype in the non-malignant MCF10A breast cell line. Furthermore, CK2β knockdown
in the MCF10A normal breast epithelial cell line resulted in a shift of FOXC2 from the
cytoplasm to nucleus even though CK2α/α′ levels were increased. The
increase in nuclear FOXC2 in these cells correlated with a mesenchymal expression pattern
and increased migration/invasion, recapitulating the phenotype of the metastatic breast
cancer cell lines. Cumulatively, these studies support the model that the ratio of
CK2α/α′:CK2β determines the phosphorylation targets of the
holoenzyme and show that FOXC2 serves as an important CK2 target for epithelial
maintenance.</p>
<p id="P23">Studies utilizing transfection of cultured epithelial cells with wild type Foxc2
have proven that over-expression of Foxc2 results in reduced levels of E-cadherin and
increased expression of mesenchymal markers such as vimentin and smooth muscle
actin.<sup><xref rid="R14" ref-type="bibr">14</xref>
</sup>
However, the specific
mechanism of this effect has been unclear since Foxc2 is increased in both the cytoplasmic
and nuclear compartments in that setting.<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
The current studies demonstrate that nuclear localization of Foxc2 is
required for expression of mesenchymal proteins and suppression of E-cadherin since
over-expression of cytoplasmically localized Foxc2-S124D fails to increase vimentin or
α-SMA protein expression or suppress E-cadherin expression. The observation that
transfection with nuclear localized Foxc2-S125L reduced the mRNA levels for E-cadherin while
increasing the message for both vimentin and α-SMA is consistent with the model that
Foxc2-mediated EMT is dependent on transcriptional regulation. Interestingly, the shift of
FOXC2 from nucleus to cytoplasm seen following re-expression of CK2β in MDA-MB-231
cells was sufficient to reduce α-SMA expression, but was not sufficient to reduce
vimentin expression or relieve the tonic inhibition of E-cadherin expression seen in these
cells. Whether this differential effect is due to the transcriptional activity of the small
amount of FOXC2 that remains in the nucleus of CK2β re-expressing MDA-MB-231 cells,
or is due to non-FOXC2 dependent regulation of vimentin and E-cadherin in these malignant
cells remains to be determined.</p>
<p id="P24">In conclusion, we show that CK2-mediated phosphorylation of Foxc2 promotes
cytoplasmic localization of Foxc2 in normal epithelial cells, and that interruption of CK2
activity by either kinase inhibition or knock-down of the enzyme results in Foxc2 nuclear
localization, increased migration, invasion, and partial loss of the epithelial phenotype.
These data suggest that the CK2 holoenzyme normally acts to maintain cells in the epithelial
state, but that this regulatory mechanism can be overcome under pathologic conditions. Thus
stimulation of CK2 dependent phosphorylation of Foxc2, or suppression of Foxc2
dephosphorylation, might serve as therapeutic targets to promote maintenance of the
epithelial phenotype and reduce the malignant phenotype in breast cancer cells.</p>
</sec>
<sec sec-type="materials|methods" id="S13"><title>MATERIALS AND METHODS</title>
<sec id="S14"><title>Cell lines and cell culture</title>
<p id="P25">Mouse proximal tubule (MPT) cells were cultured and maintained in our laboratory
as previously reported.<sup><xref rid="R16" ref-type="bibr">16</xref>
</sup>
MCF10A cells,
MDA-MB-436 and MDA-MB-231 cells were maintained by American Type Culture Collection
standards. MCF10A and MDA-MB-231 cells were a generous gift from Dr. David Rimm.
MDA-MB-436 cells were a generous gift from Dr. Marc Hansen.</p>
<p id="P26">Expression vectors and cell transfection studies Full-length murine Foxc2 (a
generous gift from Dr. Tsutomu Kume) was subcloned in-frame with the N-terminal GFP tag in
the pEGFP-C1 vector (Clontech, Mountain View, CA, USA) or the N-terminal FLAG-tag in
pFLAG-CMV (Sigma, St. Louis, MO, USA). Residues in the murine Foxc2 cDNA were mutated and
restriction sites for subcloning created by site-directed mutagenesis in the pEGFP-C1
vector according to the manufacturer’s directions. Mutations were confirmed by
sequencing at the Yale Keck Sequencing Facility. The pAB07-CK2β expression vector
was obtained from Addgene.</p>
</sec>
<sec id="S15"><title>Immunofluorescence</title>
<p id="P27">For localization studies, MPT cells were plated on glass coverslips and fed with
Opti-MEM medium (Invitrogen, Grand Island, NY, USA) supplemented with 5% FBS in
5% CO2 at 37°C. Cells were transfected with 0.8
<italic>μ</italic>
g of plasmid DNA with LIPOFECTAMINE 2000 for 4 h. Transfected
cells were stabilized for 48 h before fixation with 4% paraformaldehyde in PBS and
rinsed in PBS, prior to applying mounting solution with DAPI (Vector Laboratories,
Burlingame, CA, USA). For F-actin staining, cells were fixed with 4%
paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100, stained with
Rhodamine-phalloidin (Molecular Probes Eugene, OR, USA) and mounted with DAPI (Vector
Laboratories, Burlingame, CA, USA). Images were acquired using an inverted fluorescent
microscope with a 40x objective (Olympus) and OpenLab software (Improvision).</p>
</sec>
<sec id="S16"><title>Immunodetection</title>
<p id="P28">Immortalized MPT cells were plated in 100 mm dishes and allowed to grow in
Opti-MEM with 5% FBS for 48 h. Cells were re-fed with DMEM:F12 plus
10%FBS, then transfected with 16 <italic>μ</italic>
g of DNA. Cells were
lysed with RIPA buffer (Teknova, Hollister, CA, USA) or the NE-PER kit (Thermo Scientific,
Waltham, MA, USA) containing Halt (Thermo Scientific, Waltham, MA, USA)
protease/phosphatase inhibitors. Whole cell lysates were cleared by 10 min centrifugation
at 14000 rpm at 4<italic>°</italic>
C. Protein concentration was determined by the
Bradford assay (Bio-Rad). 50 <italic>μ</italic>
g of protein lysate was loaded on
to gels and transferred to PVDF membranes. Primary antibodies used overnight at
4<italic>°</italic>
C: anti-α-SMA (EPIT-MICS, Burlingame, CA, USA, 1184),
anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-7558), anti-E-cadherin
(BD Transduction Laboratories, San Jose, CA, USA, 610181), anti-β-actin (Novus,
Littleton, CO, USA, NB600-503), anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA;
SC-9996), anti-CK2α/α′ (Santa Cruz Biotechnology, Santa Cruz, CA,
USA; SC-136281), anti-CK2β (Pierce Biotechnology, Rockford, USA, PA5-27416) or
anti-Foxc2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-28704 or SC-21397).
Antibody–antigen complexes were identified by chemiluminesence
(ECL+System; Amersham Biosciences, Piscataway, NJ, USA).</p>
</sec>
<sec id="S17"><title>NE-PER fractionation</title>
<p id="P29">MPT cells grown in culture plates were placed on ice and fractionated using the
NE-PER kit (Thermo Scientific, Waltham, MA, USA). Cells were washed with cold PBS. Plates
were tipped on side to drain excess PBS which was aspirated off. CERI mixture was then
added to the well (200μl CERI, 2μl Halt protease/phosphatase inhibitors
per well of a 6-well plate) and vortexed vigorously for 15 seconds. Tubes were incubated
on ice for 10 min. Then 11μl of cold CERII was added to each tube and vortexed
vigorously for 10 sec. Tubes were incubated on ice 1 min, vortexed 10 sec, and centrifuged
at 13200 rpm for 5 min at 4°C. Immediately supernatant (Cytosolic Fraction) was
transferred to a clean pre-chilled 1.5ml. A quick spin was performed to remove excess
cytosolic fraction with a syringe. The pellet was washed 2 times with PBS to remove any
remaining cytosolic fraction. After the nuclear pellet was collected, the NER cocktail was
added (100μl NER, 1μl Halt protease/phosphatase inhibitors) to pellet,
incubated on ice for 10 min intervals, vortexing for 15 sec at the start of intervals for
a total of 40 min, finally centrifuging at 13200 rpm for 10 min at
4<italic>°</italic>
C. Supernatant (nuclear fraction) was transferred to a clean
pre-chilled 1.5ml tube. All fractions were stored at
−80<italic>°</italic>
C unless immunoprecipitation was performed.
Quantification of relative levels of Foxc2 in each compartment was performed using
normalization to the loading controls (LaminA/C and GAPDH) and to the proportion of total
cell protein isolated from each compartment (4.65:1, cytosolic:nuclear).</p>
</sec>
<sec id="S18"><title>Immunoprecipitation</title>
<p id="P30">Cell lysates (1000 <italic>μ</italic>
g of protein) from cells
transfected with GFP-Foxc2 were incubated with 10 <italic>μ</italic>
l of
monoclonal antibody against GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at
4°C overnight with of 20 <italic>μ</italic>
l of 50% (v/v) Protein
A/G plus–agarose bead slurry (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Beads were pelleted by centrifugation for 2 min at 1000 <italic>g</italic>
, and then
washed three times with lysis buffer, and twice with PBS. Proteins were eluted and
prepared for analysis by heating in reduced Laemmli sample buffer at 100°C for 5
min. Proteins were resolved by 4–15% gradient SDS/PAGE and transferred on
to PVDF membranes for Western blotting. Membranes were blocked with 5% (w/v)
non-fat dried milk in Tris-buffered saline, then probed either with
antibody–horseradish peroxidase conjugates containing antibody against GFP or CK2
proteins (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then horseradish peroxidase
conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA).</p>
</sec>
<sec id="S19"><title>CK2 inhibition</title>
<p id="P31">MPT cells were treated with vehicle control or 100<italic>μ</italic>
M of
TBCA (Calbiochem, Billercia, CA, USA) for 1h and subjected to cell fractionation. For
RNAi, MPT cells were transfected with scrambled siRNA (Ambion, Foster City, CA, USA),
siRNA directed to knock-down CK2α/α′ (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) or siRNA directed to knock-down CK2β (Life Technologies,
Grand Island, NY, USA) and subjected to cell fractionation.</p>
</sec>
<sec id="S20"><title>Quantitative Real-time PCR</title>
<p id="P32">Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA) and 1
mg of RNA was reverse transcribed using random hexamer primers according to the
manufacturer’s instructions (iScript; Biorad, Hercules, CA, USA). qPCR was
conducted using power SYBR green mix (SsoFast; Biorad, Hercules, CA, USA) with a iCycler
real-time PCR machine (Biorad, Hercules, CA, USA). Primer pairs were selected for their
specificity and efficiency and target gene expression levels were determined by the
comparative cycle threshold method (Ct) or ddCt (dCt of target/dCt of control) method. PCR
controls run in absence of template were consistently negative.</p>
</sec>
<sec id="S21"><title><italic>In Vitro</italic>
Kinase Assay</title>
<p id="P33">Immunoprecipitated GFP-Foxc2 or GFP-Foxc2-S124L constructs were incubated for 60
min at 30°C with and without 500U of purified protein kinase CK2 (New England
Biolabs, Ipswich, MA, USA) in the presence of kinase buffer (New England Biolabs, Ipswich,
MA, USA), 20 <italic>μ</italic>
M cold ATP (Sigma, St. Louis, MO, USA) and 2
<italic>μ</italic>
Ci of [γ-<sup>32</sup>
P]ATP (Perkin
Elmer, Waltham, MA, USA). <italic>In vitro</italic>
kinase reactions were terminated by
addition of SDS-PAGE sample buffer and boiling for 5 min. Samples were subjected to
SDS-PAGE, and the amount of <sup>32</sup>
P incorporated into GFP-Foxc2 or GFP-Foxc2-S124L
was analyzed by autoradiography, followed by immunoblot analysis.</p>
</sec>
<sec id="S22"><title>Migration Assay</title>
<p id="P34">Cell migration was determined by using a 96-well ChemoTx (Neuroprobe,
Gaithersburg, MD, USA) cell migration microplate with a pore size of 8μm. The
underside of filters was coated with 1mg/mL of collagen-I (Trevigen, Gaithersburg, MD,
USA) in PBS for 1 h at room temperature, rinsed twice in distilled water, and air dried.
Serum-starved cells were trypsinized to yield single cells. The cells were pelleted and
diluted to a final concentration of 3.3 × 10<sup>4</sup>
cells/ml in DMEM
supplemented with 1 mg of BSA/ml (DMEM/BSA) to prevent cell clumping. The bottom wells of
the ChemoTx microplate were filled with DMEM/BSA ± 10% FBS, covered with
the filter and 1000 cells added to each top well. The chamber was incubated for 12 h at
37°C in 5% CO<sub>2</sub>
. Cells adhering to the top of the filter were
removed, and cells adhering to the bottom of the filter were fixed in 100%
methanol, washed, and adherent cells stained with 1% (wt/vol) crystal violet in
20% (vol/vol) methanol for 10 min and counted visually. All experiments were
performed in quadruplicate.</p>
</sec>
<sec id="S23"><title>Invasion assay</title>
<p id="P35">Invasion assays were done using Cultrex<sup>®</sup>
96-well Basement
Membrane Extract (BME) Cell Invasion Assay (Trevigen, Gaithersburg, MD). Briefly,
triplicate transwell chambers with 8-μm pore polycarbonate filters were coated
with 50 <italic>μ</italic>
l of ice-cold 0.5× BME in 1x Coating Buffer and
incubated for one hour at 37°C. To monitor cell invasion, 5000 cells in 50
μL of serum-free media were seeded on the BME-coated filters and the lower chamber
was filled with 150 <italic>μ</italic>
l of medium ± 10% FBS as a
chemoattractant. After incubation for 24 h, the cells on the underside of the filter were
stained with crystal violent and quantified visually.</p>
</sec>
<sec id="S24" sec-type="results"><title>Statistics</title>
<p id="P36">All results are expressed as mean±s.e.m. Statistical significance was
assessed by Student’s t-test. A p-value less than 0.05 was considered to be
statistically significant.</p>
</sec>
</sec>
<sec sec-type="supplementary-material" id="S25"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="SD1"><label>1</label>
<media xlink:href="NIHMS627033-supplement-1.docx" orientation="portrait" xlink:type="simple" id="d37e619" position="anchor"></media>
</supplementary-material>
<supplementary-material content-type="local-data" id="SD2"><label>2</label>
<media xlink:href="NIHMS627033-supplement-2.tif" orientation="portrait" xlink:type="simple" id="d37e623" position="anchor"></media>
</supplementary-material>
<supplementary-material content-type="local-data" id="SD3"><label>3</label>
<media xlink:href="NIHMS627033-supplement-3.tif" orientation="portrait" xlink:type="simple" id="d37e627" position="anchor"></media>
</supplementary-material>
</sec>
</body>
<back><ack id="S26"><p>This work was supported by NIH awards to LGC (DK65109) and D.G. (DK094589).</p>
</ack>
<fn-group><fn id="FN2" fn-type="conflict"><p><bold>Conflict of Interest</bold>
</p>
<p>The authors declare no conflict of interest.</p>
</fn>
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<floats-group><fig id="F1" orientation="portrait" position="float"><label>Figure 1</label>
<caption><title>Foxc2 localization</title>
<p>(A) MPT cells, NIH3T3 cells and MPT cells transiently transfected with FLAG-Foxc2 were
subjected to cell fractionation followed by SDS-PAGE and immunoblotting for Foxc2,
LaminA/C (nuclear marker) and GAPDH (cytoplasmic compartment marker). Representative
images from an <italic>n</italic>
of 3 replicates per condition. (B) MPT cells transiently
transfected with either GFP-Foxc2, GFP-Foxc2-S124D or GFP-Foxc2-S124L were fixed and
imaged for GFP (green) and DAPI (blue). Images obtained at 40x magnification. (C) MPT
cells transiently transfected as above were subjected to cell fractionation followed by
immunoblotting with anti-GFP, anti-LaminA/C and anti-GAPDH. Representative blots from an n
of 4–6 replicates per condition.</p>
</caption>
<graphic xlink:href="nihms627033f1"></graphic>
</fig>
<fig id="F2" orientation="portrait" position="float"><label>Figure 2</label>
<caption><title>CK2 is required to maintain cytoplasmic localization of Foxc2</title>
<p>(A) MPT cells were transfected with GFP-Foxc2 and whole cell lysates immunoprecipitated
with anti-GFP or IgG isotype control and blotted for anti-GFP and anti-CK2. WCL is the
input material for the IP. (B) MPT cells were transfected with scrambled siRNA or siRNA
directed against CK2 followed 24 hours later by lysis and immunoblotting to determine
efficiency of CK2 protein reduction. Percentage knock-down determined by normalizing to
β-actin. Graph shows quantification for <italic>n</italic>
=3;
**p<0.005 relative to siRNA scrambled control. (C) MPT cells
transfected as in (B) followed by cell fractionation and immunoblotting for Foxc2,
LaminA/C and GAPDH. Graph shows quantification for <italic>n</italic>
=3;
***p<0.001, **p<0.005 relative to siRNA
scrambled control. (D) MPT cells were treated with vehicle control or
100<italic>μ</italic>
M TBCA for 1h, followed by cell fractionation and
immunoblotting for endogenous Foxc2, LaminA/C and GAPDH. Relative Foxc2 expression in each
compartment was determined as described in Methods.
<italic>n</italic>
=4;***p<0.001,
**p<0.005 relative to vehicle control. (E–F) MPT cells
transiently transfected with the indicated constructs were treated with vehicle control
(E) or 100<italic>μ</italic>
M TBCA (F) for 1h, and subjected to cell fractionation
followed by immunoblotting with anti-GFP, anti-LaminA/C and anti-GAPDH. Relative
expression was determined as described in Methods. <italic>n</italic>
=3;
**p<0.005.</p>
</caption>
<graphic xlink:href="nihms627033f2"></graphic>
</fig>
<fig id="F3" orientation="portrait" position="float"><label>Figure 3</label>
<caption><title>Serine 124 is phosphorylated <italic>in vitro</italic>
by CK2</title>
<p>MPT cells were transfected with GFP-Foxc2 or GFP-Foxc2-S124L followed by
immunoprecipitation with anti-GFP antibody and <italic>in vitro</italic>
phosphorylation
± CK2. Paired samples were subjected to SDS-PAGE and immunoblotting with anti-GFP
antibody to confirm immunoprecipitation of GFP-Foxc2 and GFP-Foxc2-S124L.</p>
</caption>
<graphic xlink:href="nihms627033f3"></graphic>
</fig>
<fig id="F4" orientation="portrait" position="float"><label>Figure 4</label>
<caption><title>Cytoplasmic localization maintains an epithelial phenotype</title>
<p>(A) MPT cells were transfected with GFP, GFP-Foxc2-S124D or GFP-Foxc2-S124L constructs x3
to achieve ~70% transfection efficiency and grown to 90% confluency. Whole
cell lysates were then blotted with the indicated antibodies. (B) Quantification of 3
experiments performed as in (A) with protein expression normalized to β-actin.
<italic>n</italic>
=3; ***p<0.001,
**p<0.005 (compared to GFP control cells). (C) Real-time PCR was
performed using RNA from cells transfected as in (A) with RNA expression normalized to
Hprt1 and MPT control. WCL=whole cell lysate. <italic>n</italic>
=3;
***p<0.001, **p<0.005 (compared to control
cells).</p>
</caption>
<graphic xlink:href="nihms627033f4"></graphic>
</fig>
<fig id="F5" orientation="portrait" position="float"><label>Figure 5</label>
<caption><title>FOXC2 nuclear expression correlates with metastatic phenotype in breast cancer cell
lines</title>
<p>(A) MCF10A, MDA-MB-436 and MDA-MB-231 whole cell lysates were subjected to immunoblotting
with anti-Foxc2 or anti-β-actin and quantified relative to β-actin.
<italic>n</italic>
=3; *p<0.05 relative to MCF10A cells. (B) MCF10A,
MDA-MB-436 and MDA-MB-231 cells were subjected to cell fractionation followed by SDS-PAGE
and immunoblotting for FOXC2, Lamin A/C and GAPDH and quantified below. Relative FOXC2
expression was determined as described in Methods. WCL=whole cell lysate,
C=Cytosolic, N=Nuclear. <italic>n</italic>
=3; *p<0.05
relative to MCF10A cells.</p>
</caption>
<graphic xlink:href="nihms627033f5"></graphic>
</fig>
<fig id="F6" orientation="portrait" position="float"><label>Figure 6</label>
<caption><title>Mesenchymal cell lines expressing nuclear FOXC2 have higher expression levels of EMT
markers and altered CK2α/α′:CK2β expression</title>
<p>(A) MCF10A, MDA-MB-436 and MDA-MB-231 whole cell lysates were subjected to immunoblotting
with the indicated antibodies. (B) Quantification of 3 experiments performed as in (A)
with protein expression normalized to β-actin loading control.
<italic>n</italic>
=3; *p<0.05 relative to MCF10A cells. (C) MPT and
MDA-MB-231 whole cell lysates (left panel) along with MPT, MCF10A and NIH3T3 cell lysates
(right panel) were subjected to immunoblotting with the indicated antibodies.
WCL=whole cell lysate.</p>
</caption>
<graphic xlink:href="nihms627033f6"></graphic>
</fig>
<fig id="F7" orientation="portrait" position="float"><label>Figure 7</label>
<caption><title>Restoration of CK2β expression in metastatic breast cancer cells promotes
FOXC2 cytoplasmic localization and reduced migration</title>
<p>(A) MDA-MB-231 cells were transfected with either an empty vector or a CK2β
expression construct x2 and grown to 90% confluency. Whole cell lysates were then
blotted with the indicated antibodies. (B) MDA-MB-231 cells transfected as in (A) were
subjected to cell fractionation and fractions immunoblotted for FOXC2, LaminA/C and GAPDH.
(C) MDA-MB-231 cells transfected as in (A) were subjected to cellular migration assays
± 10% FBS. <italic>n</italic>
=3;
***p<0.001, *p<0.05 (compared to control cells). (D)
MDA-MB-231 cells transfected as in (A) were subjected to a basement membrane extract
cellular invasion assay ± 10% FBS. <italic>n</italic>
=3;
***p<0.001, *p<0.05 (compared to control cells).
WCL=whole cell lysate, C=Cytosolic, N=Nuclear, FBS=Fetal
Bovine Serum.</p>
</caption>
<graphic xlink:href="nihms627033f7"></graphic>
</fig>
<fig id="F8" orientation="portrait" position="float"><label>Figure 8</label>
<caption><title>CK2β knockdown in normal breast epithelial cells promotes FOXC2 nuclear
localization and increased migration/invasion</title>
<p>(A) MCF10A cells were transfected with either a scrambled siRNA or siRNA directed against
CK2β and grown to 90% confluency. Whole cell lysates were then blotted
with the indicated antibodies. (B) MCF10A cells transfected as in (A) were subjected to
cell fractionation and fractions immunoblotted for FOXC2, LaminA/C and GAPDH. (C) MCF10A
cells transfected as in (A) were subjected to cellular migration assays ±
10% FBS. <italic>n</italic>
=3; ***p<0.001,
*p<0.05 (compared to control scrambled siRNA cells). (D) MCF10A cells
transfected as in (A) were subjected to a basement membrane extract cellular invasion
assay ± 10% FBS. <italic>n</italic>
=3;
***p<0.001 (compared to control cells). WCL=whole cell
lysate, C=Cytosolic, N=Nuclear, FBS=Fetal Bovine Serum.</p>
</caption>
<graphic xlink:href="nihms627033f8"></graphic>
</fig>
<fig id="F9" orientation="portrait" position="float"><label>Figure 9</label>
<caption><title>Foxc2-S124L can overcome the CK2β-mediated normalization of migration</title>
<p>(A) MCF10A cells were transfected with either empty vector, CK2β, GFP-Foxc2 or
GFP-Foxc2-S124L in the indicated combinations, grown to 90% confluency and
subjected to cell migration assays without fetal bovine serum (FBS).
<italic>n</italic>
=3; ***p<0.001 (compared to control
cells). (B) MDA-MB-231 cells transfected as in (A) were subjected to cell migration assays
without FBS. <italic>n</italic>
=3; ***p<0.001 (compared
to control cells). (C) MCF10A cells transfected as in (A) were subjected to a basement
membrane extract cellular invasion assay without FBS. <italic>n</italic>
=3;
**p<0.01, *p<0.05 (compared to control cells). (D)
MDA-MB-231 cells transfected as in (A) were subjected to a basement membrane extract
cellular invasion assay without FBS. <italic>n</italic>
=3;
**p<0.01 (compared to control cells).</p>
</caption>
<graphic xlink:href="nihms627033f9"></graphic>
</fig>
<table-wrap id="T1" position="float" orientation="portrait"><label>Table 1</label>
<caption><p>Potential CK2 site is conserved amongst Fox family members.</p>
</caption>
<table frame="void" rules="none"><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1"><graphic xlink:href="nihms627033f10"></graphic>
</td>
</tr>
</tbody>
</table>
</table-wrap>
</floats-group>
</pmc>
</record>
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