? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
p90 ribosomal S6 kinase 2 promotes invasion
and metastasis of human head and neck
squamous cell carcinoma cells
Sumin Kang,1 Shannon Elf,1 Katherine Lythgoe,1 Taro Hitosugi,1 Jack Taunton,2 Wei Zhou,1 Li Xiong,3
Dongsheng Wang,1 Susan Muller,4 Songqing Fan,1 Shi-Yong Sun,1 Adam I. Marcus,1 Ting-Lei Gu,5
Roberto D. Polakiewicz,5 Zhuo (Georgia) Chen,1 Fadlo R. Khuri,1 Dong M. Shin,1 and Jing Chen1
1Winship Cancer Institute of Emory University, Atlanta, Georgia. 2Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology,
University of California, San Francisco. 3Department of Mathematics and Computer Science and 4Department of Pathology and Laboratory Medicine,
Emory University, Atlanta, Georgia. 5Cell Signaling Technology Inc. (CST), Danvers, Massachusetts.
Metastasis continues to be the cause of more than 90% of human
cancer deaths. However, how tumors spread and kill their host
organism remains an enigma. Current underlying concepts
hypothesize that metastatic tumor cells emerge from the somat-
ic evolution of a population of cancer cells that are genetically
diversified due to selective pressures from the microenvironment.
Only a very small population of these cancer cells will achieve the
ability to colonize a distant organ when released into the circu-
lation. In addition, these metastatic cells must evade multiple
barriers that are posed by healthy tissues to successfully complete
invasion and colonization. Thus, metastasis likely represents an
evolutionary process that involves selection of genetically het-
erogeneous lineages of cancer cells within the context of a whole
organism (1, 2). Metastasis is a biological cascade of multiple
steps: loss of cellular adhesion, increased motility and invasive-
ness, entry and survival in the circulation, exit into new tissue,
and eventual colonization at a distant site. This suggests that
cells containing metastatic lesions would have to accumulate
expression of multiple, if not all, genes necessary for successful
execution of the metastatic cascade from the primary tumor (3).
Therefore, important and long-standing questions that remain
concern the identity of genes that mediate these metastasis-pro-
moting processes. Identification and characterization of these
genes will not only shed new insight into the molecular basis
of cancer metastasis but also inform therapeutic strategies to
improve the outcome of treatment of human cancers.
Head and neck squamous cell carcinoma (HNSCC) is one of the
most common types of human cancer, with an annual incidence
of more than 500,000 cases worldwide. Although recent molecular
studies have advanced our understanding of the disease and pro-
vided a rationale to develop novel therapeutic strategies, HNSCC
is still associated with severe disease- and treatment-related mor-
bidity, with a 5-year survival rate of only approximately 50%, which
has not improved in more than 30 years (4). Worse yet, the 5-year
survival rate is even lower for HNSCC patients with a single unilat-
eral LN metastasis (LNM) and less than 25% for patients with bilat-
eral LNM. Current clinical treatments of HNSCC include surgery,
radiotherapy, chemotherapy, and molecularly targeted agents. As
with most forms of cancer, treatment of HNSCC depends largely
on tumor stage. The detection of local LNM is pivotal for choosing
appropriate treatment, especially for individuals diagnosed with
HNSCC in the oral cavity or oropharynx. However, distant metas-
tasis from HNSCC to lung or bone usually represents incurable
disease. Therefore, it is of clinical interest to identify metastasis-
promoting genes in primary HNSCC tumors to improve prognosis
and define targets for therapy.
Protein kinases have been implicated in mediating prometastatic
signaling in human cancers. For example, the hERBB2 receptor
tyrosine kinase is overexpressed in 30% of cases of primary human
breast cancer, which correlates with tumor progression and poor
patient outcome (5). We began addressing this issue by examin-
ing correlations between the expression of protein kinases and
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2010;120(4):1165–1177. doi:10.1172/JCI40582.
1166? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
the tumor progression of head and neck cancer. We found that
p90 ribosomal S6 kinase 2 (RSK2) is predominantly expressed in a
spectrum of metastatic human HNSCC cell lines, compared with
the parental, poorly metastatic cells. RSK family members share
structural and functional similarities and contain 2 distinct kinase
domains, both of which are catalytically functional (reviewed in
refs. 6, 7). The C-terminal kinase domain (CTD) is responsible
for autophosphorylation at Ser386 (numbering based on the
murine RSK2 amino acid sequence) that is critical in RSK activa-
tion, whereas the N-terminal kinase domain (NTD) is believed to
phosphorylate exogenous substrates of RSK. RSK phosphorylates
multiple signaling effectors to play an essential role in a number
of cellular functions, including regulation of gene expression by
phosphorylation of transcriptional regulators, including c-Fos
and cAMP-responsive element-binding protein (CREB) (8), as well
as phosphorylation of histone H3 (9), which contributes to the
chromatin remodeling during mitosis and transcriptional activa-
tion; regulation of cell cycle by phosphorylating and inhibiting
Myt1 (10), a p34cdc2 inhibitory kinase in Xenopus extracts; and regu-
lation of cell survival by phosphorylating BAD (11), Bim (12), and
death-associated protein (DAP) kinase (13) to protect cells from
apoptosis. Therefore, RSK2 may serve as a key regulator in the
metastasis-promoting signaling network by activating multiple
signaling effectors that subsequently potentiate the invasive and
metastatic abilities of HNSCC cells.
Herein, we report that continued RSK2 expression contributes
to the maintenance of the invasive and metastatic potential of
HNSCC cells and that the pattern of RSK2 expression correlates
with HNSCC cell invasive ability as well as human head and neck
cancer progression. Moreover, we also identified multiple pro-
metastatic protein factors whose phosphorylation and activation
levels are regulated by RSK2, including the known RSK2 substrate,
CREB, and a newly identified RSK2 substrate, Hsp27. These find-
ings suggest that RSK2 programs a prometastatic signaling net-
work in HNSCC cells.
RSK2 expression correlates with cell invasive ability of diverse human
HNSCC cell lines and HNSCC progression. We performed an in vitro
Matrigel invasion assay, using diverse human HNSCC cell lines.
Based on the differential invasive ability, these cell lines were divided
into 2 groups: a poorly invasive group that includes Tu686, 37A,
Tu212, and 686LN and a highly invasive group that includes M4e,
37B, 212LN, 4A, 4B, and 886LN (Figure 1A). We found that RSK2
was overexpressed and activated, as assessed by phosphorylation
at Ser386, in the highly invasive cell lines, including M4e, 37B,
212LN, 4A, 4B, and 886LN, compared with the poorly invasive cell
IHC analysis of primary tissue samples from patients with HNSCC
Total sample no.
P = 0.007
P < 0.0001
RSK2 expression pattern correlates with HNSCC cancer progression.
IHC analysis was performed using a group of primary patient tissue
specimens, including 51 Tu–Met, 51 Tu+Met, and 50 LN+Met samples. The
P values were determined by χ2 test.
RSK2 is overexpressed in a group of highly invasive human HNSCC
metastatic cell lines, and the pattern of RSK2 expression correlates
with HNSCC cancer progression. (A) In vitro Matrigel invasion assay
demonstrates differential invasive ability of diverse human HNSCC cell
lines that are divided into 2 groups, including a poorly invasive group
that includes Tu686, 37A, Tu212, and 686LN and a highly invasive
group that includes M4e, 37B, 212LN, 4A, 4B, and 886LN (mean ± SD).
(B) RSK2 expression correlates with the invasive ability of diverse
HNSCC cells. RSK2 was overexpressed and activated in cell lines of
the highly invasive group (M4e, 37B, 212LN, 4A, 4B, and 886LN) com-
pared with cell lines in the poorly invasive group (Tu686, 37A, Tu212,
and 686LN). (C) Representative IHC staining images are shown for 0,
1+, 2+, and 3+ scores of human metastatic HNSCC tissue samples.
Original magnification, ×400.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
lines, including Tu686, 37A, Tu212, and 686LN (Figure 1B). These
data suggest that RSK2 expression may promote HNSCC cell inva-
sion and tumor metastasis.
We next conducted a study to detect RSK2 expression by an
immunohistochemistry (IHC) assay, using primary human HNSCC
patient tissue samples. We first characterized the RSK2 antibody
(Novus Biologicals) using RSK2-negative Tu212 cells and RSK2-
positive 212LN cells that were embedded in paraffin (Supplemental
Figure 1A; supplemental material available online with this article;
doi:10.1172/JCI40582DS1). Positive Western blot and IHC staining
of RSK2 was observed in 212LN cells but not in Tu212 cells. More-
over, we evaluated this antibody using primary tumor tissue samples
from xenografted mice injected with either control M4e-pLKO.1
cells or M4e-pLKO.1-RSK2 shRNA cells with stable knockdown of
RSK2 (described below). Positive Western blot and IHC staining of
RSK2 was observed in tumor tissue samples derived from control
M4e-pLKO.1 cells but not in those from M4e-pLKO.1-RSK2 shRNA
cells (Supplemental Figure 1B).
Tumor and LN specimens representing 3 categories were evalu-
ated, including primary tumors from patients with nonmetastatic
disease (Tu–Met), primary tumors from patients with metastatic
HNSCC (Tu+Met), and paired metastatic LN (LN+Met) samples from
the same patients. As shown in Figure 1C, positive staining of RSK2
was determined by the IHC signal intensity (scored as 0–3+) in the
cytoplasm in a percentage of tumor cells. Table 1 presents the sum-
marized data, with statistical analysis showing that the percent-
ages of RSK2-positive cases (scored 1+, 2+, or 3+) are significantly
higher in the paired tissue samples of primary tumor (Tu+Met) and
metastatic LNs (LN+Met) from patients with metastatic HNSCC
than the primary tumor specimens (Tu–Met) from patients with
Expression of RSK2 promotes in vitro HNSCC cell invasion. (A) Enforced expression of RSK2 resulted in increased invasive ability in poorly
metastatic HNSCC cells, including 686LN, Tu212, and 37A, in the in vitro Matrigel invasion assay. Relative invasion was normalized to the inva-
sion of control cells without transfection (mean ± SD). **P < 0.01, by 2-tailed Student’s t test. (B) Treatment with RSKI-fmk (6 μM) effectively
decreased RSK2 kinase activity, as assessed by phosphorylation level of S386, in RSK2-expressing M4e, 212LN, and 37B cells. (C) Represen-
tative areas show that inhibition of RSK2 by RSKI-fmk treatment decreased the numbers of M4e and 212LN cells that crossed the Matrigel in the
invasion assay. Original magnification, ×50. (D and E) Inhibition of RSK2 by RSKI-fmk (D) or BI-D1870 (E) (6 μM) decreased invasive ability of
M4e, 212LN, and 37B cells. Relative invasion was normalized to the invasion of control cells without drug treatment (mean ± SD; *P = 0.01–0.05).
(F) Targeting RSK2 by RSKI-fmk or BI-D1870 (6 μM) did not significantly affect the proliferation rate of M4e, 212LN, and 37B cells (mean ± SD).
Cell number of each sample was determined by normalizing the cell viability to that of a standard curve of cell number.
1168? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
nonmetastatic disease, with P values of 0.007 and less than 0.0001,
respectively. Together, these results support our hypothesis that
the RSK2 expression pattern correlates with human head and neck
cancer metastatic progression. Positive staining of RSK2 in some
cases in the group of nonmetastatic primary tumors (Tu–Met) also
suggests that RSK2 expression may already be positively selected
for, even at the primary tumor stage prior to onset of metastasis.
RSK2 promotes cell invasion in HNSCC cells. To further substanti-
ate the role of RSK2 in HNSCC metastasis, we next determined
whether RSK2 overexpression could confer invasive potential to
the poorly invasive human HNSCC cell lines, including 686LN,
Tu212, and 37A, that lack high levels of RSK2 expression. In an
in vitro Matrigel invasion assay, we observed that transiently
enforced RSK2 expression in these 3 independent cell lines (Figure
2A) significantly enhanced the invasive ability of these cells (Figure
2A). We then assessed the impact of targeting RSK2 on invasion
of HNSCC cells. We first examined the effects of inhibiting RSK2
kinase activity by using highly specific RSK inhibitors. RSK inhibi-
tor-fmk [RSKI-fmk; 1-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-
pyrrolo[2,3-d]pyrimidin-6-yl)-2-fluoroethanone] (14, 15) is a fluo-
Targeted downregulation of RSK2 attenuates HNSCC cell invasive ability. RNAi-mediated transient (A) or stable (D) knockdown of RSK2
resulted in decreased invasive ability in RSK2-expressing HNSCC cells, including M4e, 212LN, and 37B, in the in vitro Matrigel invasion assay
(B and E). Relative invasion was normalized to the invasion of control cells without RSK2 knockdown (mean ± SD). Nonsense (ns) siRNA and
RSK1 siRNA were included as negative controls in A–C; M4e cells stably infected with lentivirus carrying an empty vector pLKO.1 were included
as a negative control in D–F. *P = 0.01–0.05, **P < 0.01, by 2-tailed Student’s t test. Transient (C) or stable (F) knockdown of RSK2 did not
significantly affect the proliferation rate of M4e, 212LN, or 37B cells (mean ± SD).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
romethylketone molecule that was designed to specifically exploit
2 selectivity filters of RSK. RSKI-fmk potently inactivates the
C-terminal auto-kinase domain activity of RSK1 and RSK2 with
high specificity in mammalian cells (14). As shown in Figure 2B,
treatment with RSKI-fmk effectively inhibited RSK2 kinase activ-
ity in 3 RSK2-expressing HNSCC cell lines, including M4e, 212LN,
and 37B, as assessed by phosphorylation at Ser386, an index of
RSK2 activation. Moreover, upon RSKI-fmk treatment, these cells
demonstrated a significant attenuation of invasion (Figure 2, C
and D). Similar results were obtained by using another RSK inhibi-
tor, BI-D1870, which was derived from dihydropteridinones and
identified as a highly specific and potent inhibitor of RSK N-termi-
nal trans-kinase domain in kinase selectivity screening (Figure 2E)
(16). Thus, targeting RSK2 by 2 distinct inhibitors, which differen-
tially inhibit RSK2 kinase activity, results in marked reduction of
HNSCC cell invasive ability. However, such decreased cell invasive
ability, associated with inhibition of RSK2, was not a consequence
of reduced proliferation, because treatment with either RSKI-fmk
or BI-D1870 did not significantly affect the proliferation rate of
M4e, 212LN, and 37B cells (Figure 2F).
We also examined the effect of RNAi-mediated RSK2 knockdown
on invasion of M4e, 212LN, and 37B cells. We first used pools of
siRNA specifically targeting RSK1 or RSK2 and a non-specific
siRNA as a negative control. Both RSK1 and RSK2 siRNAs were
highly specific in decreasing their respective
target protein expression in various HNSCC
cell lines (Figure 3A). Transient transfection
of RSK2-specific siRNA induced significant
inhibition of invasion of M4e, 212LN, and
37B cells into matrix proteins in the Matri-
gel assay (Figure 3B), compared with cells
transfected with nonspecific siRNA. In con-
trast, RSK1 siRNA failed to induce inhibi-
tion of cell invasion in these HNSCC cells
(Figure 3B). Transient transfection with
nonspecific RSK1 or RSK2 siRNAs did not
significantly affect the proliferation rate of
M4e, 212LN, and 37B cells (Figure 3C; in
parallel, Western blot control is shown in
Supplemental Figure 2A). These findings
indicate that RSK2 but not RSK1 is involved
in regulation of HNSCC cell invasion. Simi-
lar results were obtained using a lentiviral
shRNA vector, pLKO.1-RSK2 shRNA, which
was documented to stably reduce RSK2 pro-
tein expression by approximately 90% in dis-
tinct HNSCC cell lines (Figure 3D). Stably
targeted downregulation of RSK2 using this
lentiviral vector in M4e, 212LN, and 37B
cells resulted in significant reduction of the
invasive ability of these cells in vitro (Figure
3E) but did not significantly affect the pro-
liferation rate of M4e, 212LN, and 37B cells
(Figure 3F; in parallel, Western blot control
is shown in Supplemental Figure 2B). An
additional control experiment showed that
stable transduction of M4e and 212LN cells
with lentiviral vectors harboring scrambled
shRNA or shRNA targeting GFP did not
significantly affect the proliferation rate of
these cells, nor did it alter the protein expression levels of RSK2 or
β-actin in M4e or 212LN cells (Supplemental Figure 3 and 4).
Targeted downregulation of RSK2 by shRNA in metastatic M4e cells inhib-
its development of LNM in a xenograft mouse model. We next assessed the
effect of knockdown of RSK2 by shRNA on the HNSCC cell meta-
static potential in vivo, using a LNM xenograft mouse model (17).
We used M4e cells that were demonstrated to be highly metastatic
in this model (17). M4e cells, which were stably transduced with the
lentiviral shRNA vector targeting RSK2 (pLKO.1-RSK2 shRNA) or
an empty control vector (pLKO.1) (Figure 4A), were injected into
nude mice. The mice were sacrificed on day 21, which was an end-
point determined based on our experience that M4e cells induced
tumor development as well as detectable LNM in 3 weeks (data not
shown). Compared with control M4e cells transduced with empty
vector, M4e-pLKO.1-RSK2 shRNA cells, with stable knockdown of
RSK2, showed a marked attenuation of LNM, which was character-
ized by the number of LNs invaded by M4e cells (Table 2), deter-
mined by IHC staining of human vimentin, a mesenchymal cell
marker that is expressed in the human metastatic M4e cells but
not in mouse LNs (Figure 4B).
In summary, these loss-of-function and gain-of-function stud-
ies in cells and mice support the view that continued RSK2 expres-
sion is important for maintaining the invasive and metastatic
potential of HNSCC cells.
RNAi-mediated stable knockdown of RSK2 significantly attenuates the metastatic potential
of M4e cells in a xenograft nude mouse model. (A) Immunoblotting results confirm the stable
knockdown of RSK2 in the M4e-pLKO.1-RSK2 shRNA cells. (B) Representative images of IHC
staining of human vimentin in mouse LNM by M4e-pLKO.1 (brown), compared with LNs from
nude mice receiving M4e-pLKO.1-RSK2 shRNA cells, to determine LNM. Original magnifica-
tion, ×40. (C) RNAi-mediated downregulation of RSK2 did not affect tumor formation of meta-
static M4e cells in vivo. Tumors from xenograft nude mice injected with control M4e-pLKO.1
cells or M4e-pLKO.1-RSK2 shRNA cells were harvested and weighed at the experimental end
point. Individual points represent differential weights of tumors from distinct mice in each group.
Average values are represented by horizontal bars. The P value was determined by 2-tailed
Student’s t test. (D) Representative images of IHC staining of Ki-67 (brown) from mice injected
with either M4e-pLKO.1 or M4e-pLKO.1-RSK2 shRNA cells. Original magnification, ×400.
1170? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
RNAi-mediated RSK2 knockdown does not attenuate proliferation and
tumor formation of metastatic HNSCC cells. We next examined the
possibility of whether the RSK2-dependent metastatic phenotype
in vivo might be a consequence of increased proliferation. In the
LNM xenograft nude mouse assay, no significant difference in the
size of primary tumors was detected between the 2 groups of mice
injected with either M4e-pLKO.1 or M4e-pLKO.1-RSK2 shRNA
cells (Figure 4C). Moreover, stable knockdown of RSK2 in M4e-
pLKO.1-RSK2 shRNA cells did not affect cell proliferation, which
was assessed using the percentage of cells with positive staining
of Ki-67 in each primary tumor tissue sample, when compared
with control M4e-pLKO.1 cells (Table 3 and Figure 4D). Together,
these in vivo data demonstrate that RSK2 possesses intrinsic pro-
metastatic activity in HNSCC cells, which is not a consequence of
RSK2 promotes metastasis in HNSCC cells by regulating multiple pro-
metastatic protein factors. To explore the molecular mechanism
underlying RSK2-enhanced metastasis, we surveyed potential
links between RSK2 and some signaling molecules of known rele-
vance to cell invasion and tumor metastasis. However, RNAi-medi-
ated RSK2 knockdown in metastatic M4e cells did not affect the
phosphorylation levels of ERK, AKT, and STAT3 (Figure 5A). To
comprehensively find mechanistic insight into the role of RSK2 in
HNSCC metastasis, we performed a phospho-proteomics–based
study using a phospho-antibody microarray (Full Moon BioSys-
tems Inc.), which provides a high-throughput platform for effi-
cient protein phosphorylation status profiling, with detection
and analysis of phosphorylation events at specific sites to identify
RSK2 downstream substrates/effectors that regulate metastasis.
The experiment was performed using the MAPK Pathway Phos-
phorylation Antibody Array, since RSK2 is a substrate of ERK.
Cell lysates obtained from M4e-pLKO.1, and M4e-pLKO.1-RSK2
shRNA cells were applied. We identified a spectrum of proteins
whose phosphorylation levels were decreased more than 15%, with
low values of 95% CI in M4e cells when RSK2 was stably knocked
down. Many of these proteins, when phosphorylated, are impor-
tant for cell migration, invasion, and tumor metastasis. These
prometastatic proteins include c-Jun, CREB, Elk-1, focal adhesion
kinase (FAK), Hsp27, IRS-1, JunB, c-MET, and Stathmin (Table 4
and Supplemental Table 1).
Among these proteins, we confirmed by immunoblotting that
targeted downregulation of RSK2 by shRNA resulted in reduced
phosphorylation and activation levels of the receptor tyrosine
kinase c-MET in M4e cells and FAK in 212LN and 37B cells (Fig-
ure 5B). Although RSK2, as a serine/threonine kinase, is unlikely to
directly regulate activation of these 2 prometastatic tyrosine kinases
by phosphorylation, a decrease in FAK and c-MET activation upon
RSK2 downregulation demonstrates the reprogramming of the
HNSCC prometastatic signaling network upon RSK2 knockdown.
RSK2 phosphorylates and activates prometastatic CREB and Hsp27. To
characterize the signaling properties of RSK2 in HNSCC cell inva-
sion, we next focused on 2 potential substrates/effectors of RSK2,
including a known RSK2 substrate, CREB, and Hsp27. RSK2 has
been demonstrated to regulate and activate CREB by phosphory-
lating Ser133 (18). CREB is a transcription factor whose signaling
is implicated in tumor growth and metastasis in human prostate
cancer (PCa) and melanoma (19–22).
Hsp27 regulates actin dynamics (23) and has been found to be
overexpressed in many human cancers. Hsp27 is regulated by phos-
phorylation at Ser15, Ser78, and Ser82. Phosphorylation of Hsp27
is associated with tumor cell migration and invasion and correlates
with LN positivity in breast cancer (24, 25). We first determined
whether RSK2 directly phosphorylates Hsp27 in an in vitro kinase
assay, in which purified recombinant Hsp27 (rHsp27) WT and
individual S15A, S78A, or S82A mutant proteins were incubated
with active recombinant RSK2 (rRSK2). As shown in Figure 5C, the
immunoblotting results using the specific phospho-Hsp27 anti-
Stable knockdown of RSK2 in M4e cells did not attenuate cell
proliferation in derived tumors
IHC staining of nuclear protein Ki-67 was performed using primary
tumor samples from each mouse. The percentage of cells with positive
staining of Ki-67 was determined. ANS; P = 0.753, by 2-tailed Student’s
t test. BKi-67+ staining average, 60.7%. CKi-67+ staining average, 63.5%.
M4e-pLKO.1-RSK2 shRNA cells, with stable knockdown
of RSK2, showed a marked attenuation of LNM
Knockdown of RSK2 led to a significant reduction of LNM, character-
ized as the number of LNs invaded by M4e cells in the xenograft nude
mice. Vimentin is a mesenchymal cell marker that is expressed in
metastatic human HNSCC cells but not in mouse LNs. Positive and
negative IHC staining of vimentin in LNs was indicated as yes (Y) and
no (N), respectively; 3 sections of each LN were analyzed. AP = 0.025,
by Fisher’s exact test.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
bodies (pS78 and pS82) demonstrate that rHsp27 WT, but not the
S78A or S82A mutants, was phosphorylated at Ser78 or Ser82 by
RSK2, respectively. In contrast, immunoblotting using a specific
phospho-Hsp27 antibody (pS15; CST) showed that both rHsp27
WT and S15A mutant proteins were not phosphorylated at Ser15
by rRSK2 (Figure 5C). Further, in vitro kinase assays using another
Hsp27 phospho-Ser15 antibody (Santa Cruz Biotechnology) and
mass spectrometry–based studies confirmed that Ser15 of Hsp27 is
not phosphorylated by RSK2 (data not shown). These data suggest
that to our knowledge Hsp27 is
a newly identified phosphoryla-
tion substrate of RSK2.
As shown in Figure 5D, CREB
S133 phosphorylation was mark-
edly reduced in metastatic M4e,
212LN, and 37B cells upon RNAi-
mediated RSK2 knockdown. Sim-
ilarly, stable RSK2 knockdown in
M4e and 212LN cells resulted
in reduced Hsp27 S78 and S82
phosphorylation. However, RSK2
knockdown did not affect phos-
phorylation levels of Hsp27 S78
and S82 in 37B cells (Figure 5, D
and E, respectively). Such differ-
ences may be due to disparities
in the cellular contexts of distinct
cell lines (discussed below).
Expression and phosphorylation lev-
els of CREB and Hsp27 are required
for RSK2-mediated pro-invasive abil-
ity of HNSCC cells. We also exam-
ined the effect of RNAi-medi-
ated knockdown of CREB and
Hsp27 on invasion of the RSK2-
expressing, metastatic M4e cells.
Both CREB and Hsp27 shRNAs
were specific in decreasing their
respective target protein expres-
sion in M4e cells (Figure 6A).
Stably targeted downregulation
of CREB or Hsp27, using lenti-
viral vectors containing distinct
shRNAs in M4e cells, resulted in
significant reduction of invasive
ability in vitro (Figure 6B) but
did not significantly affect the
proliferation rate of these cells
We also found that enforced
expression of RSK2 enhanced
the invasive ability of Tu212
cells, whereas shRNA-mediated
knockdown of CREB (Figure
6D) significantly attenuated
such RSK2 expression–induced
cell invasion. Moreover, stable
expression of CREB WT result-
ed in significantly increased cell
invasive ability of 686LN (Figure
6E) and Tu212 (Figure 6F) cells, and cells expressing a phospho-
mimetic mutant CREB S133D demonstrated further increased cell
invasive ability. In contrast, overexpressing the phospho-deficient
mutant CREB S133A similarly resulted in increased cell invasion of
686LN and Tu212 cells, as did CREB WT (Figure 6, E and F, respec-
tively), compared with control cells, but failed to further enhance
the cell invasive ability like the phospho-mimetic mutants. These
results suggest that expression of CREB has both S133 phosphory-
lation-dependent and -independent effects on HNSCC cell inva-
Targeting RSK2 by shRNA leads to decreased phosphorylation levels of multiple prometastatic protein
factors, including tyrosine kinases, c-MET and FAK, and RSK2 phosphorylation substrates, CREB and
Hsp27. (A) Stable knockdown of RSK2 did not affect phosphorylation and activation levels of ERK, AKT,
and STAT3. Immunoblotting was performed using cell lysates obtained from M4e-pLKO.1 or M4e-pLKO.1-
RSK2 shRNA cells. (B) Immunoblotting results demonstrate that targeted downregulation of RSK2 resulted
in decreased tyrosine phosphorylation and activation of prometastatic tyrosine kinases c-MET in M4e
cells and FAK in 212LN and 37B cells. β-actin was detected as a loading control. (C) RSK2 directly
phosphorylated Hsp27 at S78 and S82 but not S15. Purified rHsp27 WT and individual S15A, S78A, or
S82A mutant proteins were incubated with active rRSK2 in an in vitro kinase assay. Phosphorylation at
Ser15, Ser78, and Ser82 in rHsp27 was detected by specific antibodies phospho-Hsp27 pS15, pS78, and
pS82. (D) RNAi-mediated knockdown of RSK2 resulted in reduction in phosphorylation levels of CREB in
RSK2-expressing M4e, 212LN, and 37B HNSCC cells and decreased Hsp27 S78 phosphorylation in M4e
and 212LN cells but not 37B cells. (E) RNAi-mediated knockdown of RSK2 resulted in reduction in the
phosphorylation level of Hsp27 S82 in M4e and 212LN cells but not 37B cells.
1172? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
sion. In poorly invasive 686LN and Tu212 cells that lack high levels
of RSK2 expression, only the phospho-mimetic CREB S133D, but
not the phospho-deficient CREB S133A mutant, further increased
cell invasive ability, compared with CREB WT. This suggests that
the S133 phosphorylation-dependent pro-invasive effects of CREB
depend on RSK2 expression and activation.
RSK2 promotes cell invasion by phosphorylating Hsp27 to regulate stabi-
lization of actin filaments in HNSCC cells. Similarly, shRNA-mediated
knockdown of Hsp27 significantly attenuated Tu212 cell inva-
sion conferred by enforced expression of RSK2 (Figure 7A). Stable
expression of Hsp27 WT led to increased cell invasion of poorly
invasive Tu212 cells (Figure 7B) but not 686LN cells (Figure 7B).
Interestingly, expression of Hsp27 phospho-mimetic mutants
S78D or S82D did not lead to enhanced cell invasive capability
compared with cells expressing Hsp27 WT, whereas expression
of the double-mutant Hsp27 S78D/S82D significantly potenti-
ated cell invasion of Tu212 (Figure 7B) and 686LN (Figure 7B).
This result demonstrates that phosphorylation of Hsp27 at both
Ser78 and Ser82 by RSK2 is crucial for its proinvasive function in
HNSCC cells, while phosphorylation of Ser78 or Ser82 alone is
insufficient to activate Hsp27. In contrast, overexpression of the
phospho-deficient mutants Hsp27 S78A/S82A similarly resulted
in increased cell invasion of 686LN and Tu212 cells to a simi-
lar degree as Hsp27 WT cells, compared with control cells, but
failed to further enhance the cell invasive ability, like the phos-
pho-mimetic mutant Hsp27 S78D/S82D (Figure 7B). Thus, these
results together also implicate that Hsp27 has both S78/S82 phos-
phorylation-dependent and -independent effects on HNSCC cell
invasion. In poorly invasive Tu212 cells that lack high expression
levels of RSK2, only the phospho-mimetic double-mutant Hsp27
S78D/S82D, but not the single mutants S78D and S82D or the
phospho-deficient S78A/S82A mutant, further increased cell
invasive ability compared with Hsp27 WT (Figure 7B). This sug-
gests that the S78/S82 phosphorylation-
dependent proinvasive effects of Hsp27
depend on RSK2 expression and activa-
tion. However, in 686LN cells, Hsp27 may
only have the S78/S82 phosphorylation-
dependent effect because overexpression
of Hsp27 WT in these cells did not result
in increased cell invasion compared with
control cells (Figure 7B).
We also found that stable expression
of the Hsp27 phospho-mimetic mutant
S78D/S82D, but not WT or the phospho-
deficient S78A/S82A mutant, partially res-
cued cell invasion in M4e cells with stable
knockdown of RSK2 (Figure 7C). However,
neither RNAi-mediated stable knockdown
of RSK2 nor stable expression of Hsp27
WT, S78D/S82D, or S78A/S82A variants
significantly affected the proliferation rate
of M4e cells (Supplemental Figure 5).
Hsp27 has been implicated in regula-
tion of cytoskeleton dynamics by stabiliz-
ing actin filaments (26, 27). To determine
whether RSK2 regulates actin filaments,
we examined the integrity of actin fila-
ments in 212LN and M4e cells with stable
knockdown of RSK2. We found that actin
filaments were well organized and distributed evenly throughout
the control 212LN and M4e cells transduced with empty vector.
In contrast, knockdown of RSK2 induced the disruption of actin
filaments and redistribution of actin to the cell membrane (Figure
7D). However, stable expression of the Hsp27 phospho-mimetic
mutant S78D/S82D, but not WT or the phospho-deficient S78A/
S82A mutant, rescued the formation of actin filaments in M4e
cells with stable knockdown of RSK2 (Figure 7D), which correlates
with rescued M4e cell invasion, as shown in Figure 7C. These data
demonstrate that RSK2 regulates stabilization of actin filaments
in HNSCC cells through phosphorylation of Hsp27.
Our findings suggest that RSK2 protein expression pattern corre-
lates with the invasive ability of diverse human HNSCC cell lines,
as well as human head and neck cancer progression. Continued
RSK2 expression contributes to the maintenance of the invasive
and metastatic potential in HNSCC cells in vitro and in vivo,
respectively. IHC study of human primary tissue samples revealed
that 49% of primary tumor and 62% of LN tissue samples from
patients with metastatic HNSCC were positive for RSK2 staining,
which suggests that RSK2 overexpression might contribute to
metastasis in a large subset of HNSCC cases. However, this find-
ing also warrants further study to identify alternative signaling
pathways that stimulate cell invasion and tumor metastasis in the
We also identified multiple prometastatic proteins whose phos-
phorylation and activation levels are regulated by RSK2 in HNSCC
cells, using a phospho-antibody microarray–based proteomics
approach, which has the unique capability of quantitative profiling
of protein phosphorylation levels, by using paired unphospho- and
phospho-antibodies for each protein. Moreover, this approach is
helpful to not only identify potential substrates of particular pro-
Protein factors whose phosphorylation states decreased in M4e cells when RSK2 was
stably knocked down by shRNA
The phospho-antibody microarray identified a list of protein factors whose phosphorylation states
decreased in M4e cells when RSK2 was stably knocked down by shRNA. The signal intensities
of phosphorylated proteins and the total protein levels were determined. The ratio of each protein
was determined as the ratio between the percentages of phosphorylated proteins in total proteins
in M4e-pLKO.1-RSK2 shRNA and M4e-pLKO.1 cells. 95% CI was determined to demonstrate the
significance of the signal alteration for each protein factor. Tested RSK2 substrates in the current
report are indicated by the √ symbol.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
tein kinases, but also reveal connections among “remote” signaling
pathways. For example, the phospho-antibody microarray–based
studies revealed that phosphorylation and activation levels of 2
tyrosine kinases, c-MET and FAK, decreased upon RNAi-mediated
knockdown of RSK2 in metastatic M4e cells. FAK (phospho-Y861
antibody included in the microarray) localizes at the sites of integ-
rin clustering and cell attachment to the extracellular matrix and
is central to the processes of cells migration and invasion. FAK is
activated through either intrinsic autophosphorylation in response
to stimuli, such as integrin engagement, or transphosphorylation
by receptor tyrosine kinases (Y397) and Src family kinases (Y576,
Y577, and Y861) (28, 29). Receptor tyrosine kinase c-MET proteins
form heterodimers and function as receptors of HGF. Phosphory-
lation of Y1234/Y1235 in the juxtamembrane domain (phospho-
antibodies included in the microarray) has been demonstrated to
be essential for c-MET kinase activity and oncogenic potential (30,
31). Transgenic mice for either c-MET or HGF develop metastatic
tumors (32), and NIH 3T3 cells overexpressing either c-MET or
HGF are tumorigenic, with the derived tumors being extremely
metastatic in xenograft nude mice (33). Our finding that knock-
down of RSK2 resulted in decreased FAK and c-MET phosphoryla-
tion and activation suggests a crosstalk between signaling pathways
involving these 2 prometastatic tyrosine kinases and the serine/
threonine kinase RSK2, one which is unlikely to directly regulate
phosphorylation and activation of c-MET and FAK. Therefore, our
phospho-antibody microarray–based studies have paved the way
for new discoveries of novel signaling pathways/crosstalk to medi-
ate RSK2-dependent prometastatic signals and provide a fairly
comprehensive view of the molecular and cellular events associated
with HNSCC metastasis.
CREB is a transcription factor whose signaling is implicated in
promoting tumor progression, stimulating growth, conferring
apoptotic resistance, and supporting angiogenesis. In human PCa,
CREB is associated with androgen-independent progression and
promotes PCa bone metastasis (34). In human melanoma cells,
CREB appears to be a mediator of tumor growth and metastasis,
and the expression of a dominant-negative form of CREB sensi-
tizes melanoma cells to apoptosis and inhibits their growth and
metastasis (19–22). RSK2 has been implicated to regulate CREB
by phosphorylating Ser133. Phosphorylation at Ser133 dictates
the ability of CREB to interact with the coactivator CREB-bind-
ing protein (CBP), which mediates functional contacts with the
basal transcriptional machinery (18). Our findings suggest that
a transcription-dependent signaling cascade — RSK2 → CREB →
prometastatic or antimetastatic genes — may be a key component
of the RSK2 prometastatic signaling network in HNSCC cells.
Unlike the high-molecular-weight Hsps, Hsp70 and Hsp90,
which promote protein folding, oligomerization, and translo-
cation, Hsp27 regulates actin dynamics and apoptosis (23, 35).
Hsp27 has been found to be overexpressed in breast cancer, PCa,
gastric cancer, ovarian cancer, and urinary bladder cancer, and its
overexpression is associated with aggressive tumor behavior and
poor survival rate as well as resistance to chemotherapy (36, 37).
Hsp27 activity is regulated by phosphorylation at Ser15, Ser78,
and Ser82, which induces redistribution of the large oligomers
into small tetrameric units (38) and Hsp27 nuclear translocation
to prevent apoptosis (39). Recently, it was reported that attenu-
ation of Hsp27 phosphorylation by the specific microtubule
inhibitor KRIBB3 results in inhibition of tumor cell migration
and invasion (25), while enhanced phosphorylation at Ser78 of
Hsp27 significantly correlates with HER-2/neu and LN positivity
in breast cancer (24). Our findings demonstrate that RSK2 signals
through Hsp27 to regulate actin filaments and cell invasion via
direct phosphorylation of Hsp27 at both Ser78 and Ser82 but not
RSK2 promotes HNSCC cell invasion
through phosphorylation and activation
of the downstream substrate CREB. (A)
RNAi-mediated stable knockdown of
CREB and Hsp27 in M4e cells. (B) Knock-
down of CREB and Hsp27 significantly
reduced the invasive ability of metastatic
M4e cells. Relative invasion was normal-
ized to the invasion of M4e-pLKO.1 cells
(mean ± SD; *P = 0.01–0.05). (C) Stable
knockdown of CREB and Hsp27 did not
significantly affect the proliferation rate of
M4e cells (mean ± SD). (D) Expression of
RSK2 promoted invasive ability of Tu212
cells, whereas targeted downregulation
of CREB significantly attenuated RSK2-
dependent invasion. Relative invasion was
normalized to the invasion of control Tu212
cells (mean ± SD; **P < 0.01). (E and F)
Stable expression of the phospho-mimetic
CREB S133D mutant but not the phospho-
deficient S133A mutant, further significant-
ly enhanced 686LN (E) and Tu212 (F) cell
invasion, compared with CREB WT. Rela-
tive invasion was normalized to the inva-
sion of control cells harboring an empty
vector (mean ± SD; *P = 0.01–0.05).
1174? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
Ser15. This may represent a transcription-independent mechanism
underlying RSK2-mediated prometastatic signaling in HNSCC
cells, in addition to the proposed transcription-dependent mecha-
nism involving CREB.
The differential phosphorylation levels of Hsp27 in distinct met-
astatic HNSCC cell lines (Figure 5, D and E) indicate that each can-
cer cell line provides a unique platform for laboratory research. Dif-
ferent cellular contexts among various tumor cell lines, even those
RSK2 promotes stabilization of actin filaments in HNSCC cells through phosphorylation and activation of Hsp27. (A) RNAi-mediated knockdown
of Hsp27 significantly attenuated Tu212 cell invasion conferred by exogenous expression of RSK2. Relative invasion was normalized to the
invasion of control Tu212 cells (mean ± SD; **P < 0.01). (B) Stable expression of the phospho-mimetic Hsp27 S78D/S82D double mutant, but
not the phospho-deficient Hsp27 S78A/S82A mutant or the Hsp27 S78D and Hsp27 S82D single mutants, led to further significantly enhanced
invasion of poorly invasive Tu212 and 686LN cells, compared with Hsp27 WT. Relative invasion was normalized to the invasion of control cells
harboring an empty vector (mean ± SD; *P = 0.01–0.05; **P < 0.01). (C) Stable expression of Hsp27 S78D/S82D mutant, but not WT or S78A/
S82A mutant, rescued the cell invasion attenuated by stable knockdown of RSK2 in M4e cells. Relative invasion was normalized to the invasion
of M4e-pLKO.1 cells (mean ± SD; *P = 0.01–0.05). (D) Actin immunofluorescent staining shows that RNAi-mediated stable knockdown of RSK2
resulted in disruption of actin filaments in 212LN and M4e cells, whereas stable expression of the Hsp27 phospho-mimetic mutant S78D/S82D,
but not WT or the phospho-deficient S78A/S82A mutant, rescued the formation of actin filaments. Cells were fixed and stained with phalloidin
conjugated with Alexa Fluor 555. The integrity of actin filaments was analyzed by confocal microscopy. Original magnification, ×1,000.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
derived from human cancer patients with the same disease symp-
toms and diagnosis, may require different molecular mechanisms
for tumorigenesis and metastasis. Since targeting RSK2 attenuated
the cell invasive ability of 37B cells, RSK2 may not signal through
Hsp27 to promote cell invasion in 37B cells, as it does in M4e and
212LN cells. This also suggests that an alternative kinase or kinas-
es may exist to phosphorylate Hsp27 S78 and S82 in 37B cells in
the absence of RSK2. Interestingly, forced overexpression of either
CREB WT or Hsp27 WT in poorly invasive HNSCC cells enhances
cell invasion, and the phosphorylation-dependent contribution
is modest compared with the effects of the nonphosphorylatable
constructs in several contexts. This also suggests that different
molecular mechanisms may be required by tumor cells with differ-
ent cellular contexts to mediate proinvasive signals.
We recently demonstrated that targeting RSK2 by siRNA or
the highly specific RSK inhibitor RSKI-fmk effectively induced
apoptotic cell death in FGFR3-expressing, human t(4;14) mul-
tiple myeloma cell lines and primary patient myeloma cells, with
minimal nonspecific cytotoxicity in human cells (40). These data
provide proof of principle that not only suggest the therapeutic
potential of targeting RSK2 in related human malignancies but
also demonstrate that RSK inhibitors, such as RSKI-fmk, may
have acceptable side effects. Furthermore, we found that RSK2
is more likely to be involved in HNSCC tumor metastasis than
tumor initiation and growth in a xenograft mouse model (Table 2
and Figure 4). Therefore, RSK2 may represent an attractive target
to attenuate tumor invasion and widespread metastasis, but not
necessarily to induce regression of the primary tumor. This find-
ing warrants the development of combined therapeutic strategies
to treat metastatic HNSCC using RSK2 antagonists and other
anticancer reagents that abrogate tumor growth and progression.
Together, our studies not only advance the molecular understand-
ing of tumor progression and metastasis but also inform novel
therapies to treat metastatic HNSCC.
Reagents. The RSK-specific inhibitor RSKI-fmk was described previously
(14, 15). BI-D1870 was purchased from the University of Dundee, Scot-
land, United Kingdom. siRNA was ordered from Dharmacon. Lentiviral
shRNA vectors targeting RSK2, CREB, and Hsp27 were purchased from
Open Biosystems. The RSK2 construct has been previously described (40).
pET53-DEST-Hsp27 variants were generated for bacterial recombinant
protein purification. Recombinant active RSK2 was from Invitrogen.
Myc-CREB and Myc-Hsp27 variants were subcloned into retroviral vector
pMSCV-hyg. Various mutants were generated using the QuikChange-XL
Site-Directed Mutagenesis Kit (Stratagene).
Cell culture, lentiviral infection, retroviral infection, and proliferation assay.
HNSCC Tu686 and 686LN cell lines were provided by Peter G. Sacks, New
York University College of Dentistry, New York, New York. Tu212 and
212LN were provided by Gary L. Clayman, University of Texas MD Ander-
son Cancer Center, Houston. The 4A, 4B, 37A, and 37B cell lines were
provided by Theresa L. Whiteside, University of Pittsburgh, Pennsylvania.
M4e was described previously (17). All HNSCC cell lines were cultured in
DMEM/Ham’s F-12 50/50 mix medium in presence of 10% FBS. HNSCC
cell lines stably expressing lentiviral shRNA were cultured in presence of
puromycin (2 μg/ml). Lentivirus stocks carrying shRNA were generated
by transfecting 293T cells with 3 μg of lentiviral vector encoding shRNA,
3 μg of pHRCMV8.2ΔR, and 0.3 μg CMV-VSVG, using Lipofectamine 2000
(Invitrogen). Forty-eight hours after transfection, supernatant harboring
lentiviruses were collected. HNSCC cells were infected by lentiviruses in
6-well plates by applying infection cocktail (1 ml growing media, 0.34 ml
of virus stock, and 15 μg of polybrene). After 48 hours, infected cells were
selected using 2 μg/ml of puromycin for a week. Retrovirus production and
retroviral infection were performed as previously described (40). For prolif-
eration assays, 1 × 104 cells were seeded into 96-well plates, and the relative
cell viability at each experimental time point was determined by using the
Celltiter96AQueous One Solution Proliferation Kit (Promega). A standard
curve of the cell number was generated, and the cell number of each sample
was determined by normalizing the cell viability to that of the curve.
Antibodies. RSK1, RSK2, and phospho-Hsp27 (pS15) antibodies were
from Santa Cruz Biotechnology; specific antibodies against phospho-RSK
(S380), p44/42 ERK, phospho-p44/42 ERK (T202/T204), AKT, phospho-
AKT (S473), STAT3, phospho-STAT3 (Y705), MET, phospho-MET (Y1234/
Y1235), CREB, phospho-CREB (S133), Hsp27, phospho-Hsp27 (S15, S78,
and S82), Myc, and FAK were from CST; phospho-FAK (Y397) was from
Invitrogen; antibodies against GST and β-actin were from Sigma-Aldrich.
Anti-RSK2 antibody from Novus Biologicals, prediluted anti–Ki-67 anti-
body from Invitrogen, and anti-human vimentin antibody from Santa
Cruz Biotechnology were used for immunohistochemical staining.
Purification of rHsp27 proteins and in vitro kinase assay. (His)6-tagged Hsp27
proteins were purified by sonication of BL21(DE3)pLysS cells obtained
from 250 ml of culture with 0.5 mM IPTG induction for 6 hours at 25°C.
Cellular lysates were resolved by centrifugation and loaded onto a Ni-NTA
column (Qiagen) in 20 mM imidazole. After a step of washing twice, the
protein was eluted with 250 mM imidazole. Proteins were desalted on a
PD-10 column (GE Healthcare Life Sciences) and the purification effi-
ciency was examined by Coomassie blue staining and Western blotting.
To determine the ability of RSK2 to phosphorylate Hsp27, 150 ng purified
recombinant (His)6-Hsp27 WT and S15A, S78A, or S82A were incubated
with 500 ng recombinant active RSK2 in 20 mM MOPS, 5 mM EGTA,
1 mM DTT, 25 mM β-glycerol phosphate, 1 mM Na3VO4, and 15 mM
MgCl2 along with 10 mM MgAc and 0.1 mM ATP for 30 minutes at 30°C.
Phosphorylation of Ser15, Ser78, and Ser82 of Hsp27 was detected by cor-
responding specific phospho-antibodies.
In vitro cell invasion assay. In brief, transwell inserts with 8-μm pores
(BD Biosciences) were coated with Matrigel (272 μg/ml). Approximately
2 × 105 HNSCC cells were seeded in the upper chambers in 300 μl serum-
free medium, while 500 μl of medium supplemented with 10% FBS as a
chemoattractant was placed in the lower wells. The chambers were incu-
bated at 37°C in a CO2 incubator. After 48 hours, the chambers were
pulled out, and the noninvading cells on the upper surface were removed
with the cotton swab. The cells that invaded to the lower surface of the
membrane were fixed in methanol, air dried, and stained with 0.1% crys-
tal violet for 10 minutes. The stained cells were counted in 3 random
fields (at ×200 magnification) using a light microscope. The tumor cell
invasion was assessed as the number of cells that had passed through the
Immunohistochemical staining. Archived, formalin-fixed, paraffin-embed-
ded tumor specimens from HNSCC patients (prior to April 2003) that
had given informed consent were identified from pathology files at the
Department of Pathology and Laboratory Medicine, Emory University
Hospital. Approval for use and care of these specimens was given from the
Institutional Review Board of Emory University School of Medicine. Clini-
cal information for the patients was obtained from the pathology files at
Emory University Hospital under the guidelines and with approval from
the Institutional Review Board of Emory University School of Medicine
and according to the Health Insurance Portability and Accountability Act.
Only tumors from patients that were not treated previously with either
chemotherapy or radiation therapy were used. After deparaffinization
and rehydration, human tissue sections were incubated in 3% hydrogen
1176? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
peroxide to suppress endogenous peroxidase activity. Antigen retrieval
was achieved by microwaving the sections in 100 mM Tris (pH 10.0). Sec-
tions were then blocked by incubation in 2.5% horse serum. The primary
antibodies and monoclonal mouse anti-RSK2 antibody (Novus Biologi-
cals) were applied to the slides at a dilution of 1:500 and incubated at 4°C
overnight. Detection was achieved with the Avidin-Biotin Complex Sys-
tem (Vector Laboratories). Slides were stained with 3,3′-diaminobenzidine,
washed, counterstained with hematoxylin, dehydrated, treated with xylene,
and mounted. Immunohistochemical staining results were reviewed and
scored as follows: 0, no staining and no background; 1+, weak cytoplas-
mic staining in more than 30% of cells; 2+, moderately intense cytoplasmic
staining in more than 30% of cells but without intense staining in some
cells; and 3+, cytoplasmic staining in more than 30% of tumor cells with
markedly intense cytoplasmic staining. A similar staining procedure was
performed to stain human vimentin, using LN sections of xenografted
mice, and to stain Ki-67, using primary tumor tissue samples from these
mice. Sodium citrate buffer (10 mM) was used for antigen retrieval, and
monoclonal mouse anti-vimentin antibody was used at a dilution of 1:500
for human vimentin staining.
Xenograft nude mouse assay. Based on protocols approved by the Institu-
tional Animal Care and Use Committee of Emory University, nude mice
(athymic nu/nu) aged 4–6 weeks from Taconic were divided into 2 groups
with similar average of weight. Each mouse was injected with 0.5 × 106
M4e-pLKO.1 or M4e-pLKO.1-RSK2 shRNA cells suspended in 50 μl of PBS
into the submandibular to mylohyoid muscle as described previously (17).
Tumor formation was assessed every 2–3 days. Tumor growth was recorded
by measurement of 2 perpendicular diameters of the tumors over a 3-week
course, using the formula 4π/3 × (width/2)2 × (length/2). Mice were sacri-
ficed 3 weeks after injection, and cervical LNs and tumors were collected,
fixed immediately in 10% formalin, and embedded in paraffin. Tissue
sections of LNs were stained with hematoxylin–eosin and anti-human
vimentin antibody, and the primary tumor tissue sections were stained
with hematoxylin–eosin and anti-RSK2 or anti-human Ki-67 antibodies.
Metastases to LNs were identified by positive human vimentin staining,
which is a mesenchymal cell marker that is expressed in metastatic human
HNSCC cells but not in mouse LNs.
Phospho-protein profiling by phospho-antibody array. The experiment was
performed by Full Moon BioSystems Inc. Cell lysates obtained from M4e-
pLKO.1 and M4e-pLKO.1-RSK2 shRNA cells were applied to the MAPK
Pathway Phosphorylation Antibody Array that was designed and manu-
factured by Full Moon Biosystems Inc. The array contains 185 antibod-
ies, each of which has 6 replicates that are printed on standard-size coated
glass microscope slides. In brief, 100 μg of cell lysates in 50 μl of reaction
mixture were labeled with 1.43 μl of biotin in 10 μg/μl N,N-dimethyfor-
mamide. The resulting biotin-labeled proteins were diluted 1:20 in Cou-
pling Solution (Full Moon Biosystems Inc.) before being applied to the
array for conjugation. The Antibody Microarray was first blocked with
blocking solution for 30 minutes at room temperature, rinsed with Milli-Q
grade water for 3 minutes, and dried with compressed nitrogen, followed
by incubation with the biotin-labeled cell lysates at 4°C overnight. The
array slides were washed 3 times with 60 ml of 1X Wash Solution (Full
Moon Biosystems Inc.) for 10 minutes each, the conjugated labeled pro-
teins were detected using Cy3-conjugated streptavidin. For each antibody,
we computed the following phosphorylation ratio (phosphorylated and
matching unphosphorylated values are denoted by phospho and unphos-
pho in both the control data and experiment data):
A 95% CI was used to quantify the precision of the phosphorylation
ratio based on the analysis of the replicates. A web-based program for
conducting the data analysis and generating the result table is available
at http://www.mathcs.emory.edu/panda. Detailed description is available
in Supplemental Methods.
Immunofluorescence staining and microscopy. For F-actin labeling, cells were
seeded on glass coverslips, fixed in PHEMO buffer (68 mmol/l PIPES,
25 mmol/l HEPES, 15 mmol/l EGTA, and 3 mmol/l MgCl2) with 3.7% form-
aldehyde, 0.05% glutaraldehyde, and 0.5% Triton X-100 for 10 minutes. Cells
were washed in PBS and then blocked in 10% goat serum for 10 minutes.
F-actin was stained for 45 minutes with Alexa Fluor 555–conjugated phal-
loidin (5 U/ml; Invitrogen) in PBS containing 5% goat serum. The cover-
slips were washed in PBS, mounted, and imaged on a Zeiss LSM 510 META
confocal microscope. Confocal z-sections were acquired using a ×100 Zeiss
Plan-Apo (numerical aperture, 1.4) oil objective, with identical acquisition
parameters (laser intensity, gain, and zoom) in all experimental groups.
The z-distance between each optical section was on average 500 nm. Images
were collected in 8-bit format, with 1,024 × 1,024 resolution, and maximum
projection images were created from the z-stacks. Images were exported to
Adobe Photoshop, and contrast levels were expanded in all images.
Statistics. Statistical analysis and graphical presentation were done using
GraphPad Prism 4.0. Data shown are from 1 experiment that is representa-
tive of multiple independent experiments and are given as mean ± SD. Sta-
tistical analysis of significance (P values) was based on the χ2 test for Table 1,
Fisher’s exact test for Table 2, and 2-tailed Student’s t test for all the other
figures and tables. P values of less than 0.05 were considered significant.
We gratefully acknowledge the critical reading of the manuscript
by Benjamin H. Lee and the technical support during the xenograft
experiments by Ling Su. We thank Laura Bender at the microscope
core facility at the Winship Cancer Institute of Emory University
for the expert technical assistance. This work was supported in
part by NIH grant CA120272 (to J. Chen) and Head and Neck Can-
cer SPORE grant P50CA128613 (to S. Kang, S. Muller, S.-Y. Sun,
Z.G. Chen, F.R. Khuri, D.M. Shin, and J. Chen). S. Kang is a Special
Fellow of the Leukemia and Lymphoma Society. T. Hitosugi is a
Fellow Scholar of the American Society of Hematology. J. Chen,
S.-Y. Sun, Z.G. Chen, F.R. Khuri, and D.M. Shin are Georgia Cancer
Coalition Distinguished Cancer Scholars. J. Chen is an American
Cancer Society Basic Research Scholar.
Received for publication July 21, 2009, and accepted in revised
form January 13, 2010.
Address correspondence to: Sumin Kang or Jing Chen, Winship
Cancer Institute of Emory University, 1365-C Clifton Road NE,
Atlanta, GA 30322. Phone: 404.778.1880; Fax: 404.778.4755;
E-mail: firstname.lastname@example.org (S. Kang). Phone: 404.778.5274; Fax:
404.778.5520; E-mail: email@example.com (J. Chen).
1. Gupta GP, Massague J. Cancer metastasis: building
a framework. Cell. 2006;127(4):679–695.
2. Nguyen DX, Massague J. Genetic determinants of
cancer metastasis. Nat Rev Genet. 2007;8(5):341–352.
3. Fidler IJ. The pathogenesis of cancer metastasis: the
‘seed and soil’ hypothesis revisited. Nat Rev Cancer.
4. Haddad RI, Shin DM. Recent advances in head and
neck cancer. N Engl J Med. 2008;359(11):1143–1154.
5. Ursini-Siegel J, Schade B, Cardiff RD, Muller WJ.
Insights from transgenic mouse models of ERBB2-
induced breast cancer. Nat Rev Cancer. 2007;
research article Download full-text
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
6. Blenis J. Signal transduction via the MAP kinases:
proceed at your own RSK. Proc Natl Acad Sci U S A.
7. Frodin M, Gammeltoft S. Role and regulation of 90
kDa ribosomal S6 kinase (RSK) in signal transduction.
Mol Cell Endocrinol. 1999;151(1–2):65–77.
8. Buck M, Poli V, Hunter T, Chojkier M. C/EBPbeta
phosphorylation by RSK creates a functional XEXD
caspase inhibitory box critical for cell survival. Mol
9. He Z, Ma WY, Liu G, Zhang Y, Bode AM, Dong Z.
Arsenite-induced phosphorylation of histone H3 at
serine 10 is mediated by Akt1, extracellular signal-
regulated kinase 2, and p90 ribosomal S6 kinase 2
but not mitogen- and stress-activated protein
kinase 1. J Biol Chem. 2003;278(12):10588–10593.
10. Palmer A, Gavin AC, Nebreda AR. A link between
MAP kinase and p34(cdc2)/cyclin B during oocyte
maturation: p90(rsk) phosphorylates and inacti-
vates the p34(cdc2) inhibitory kinase Myt1. EMBO J.
11. Shimamura A, Ballif BA, Richards SA, Blenis J.
Rsk1 mediates a MEK-MAP kinase cell survival sig-
nal. Curr Biol. 2000;10(3):127–135.
12. Dehan E, et al. betaTrCP- and Rsk1/2-mediated deg-
radation of BimEL inhibits apoptosis. Mol Cell. 2009;
13. Anjum R, Roux PP, Ballif BA, Gygi SP, Blenis J.
The tumor suppressor DAP kinase is a target of
RSK-mediated survival signaling. Curr Biol. 2005;
14. Cohen MS, Zhang C, Shokat KM, Taunton J.
Structural bioinformatics-based design of selec-
tive, irreversible kinase inhibitors. Science. 2005;
15. Cohen MS, Hadjivassiliou H, Taunton J. A clickable
inhibitor reveals context-dependent autoactivation
of p90 RSK. Nat Chem Biol. 2007;3(3):156–160.
16. Sapkota GP, et al. BI-D1870 is a specific inhibitor
of the p90 RSK (ribosomal S6 kinase) isoforms in
vitro and in vivo. Biochem J. 2007;401(1):29–38.
17. Zhang X, et al. A lymph node metastatic mouse
model reveals alterations of metastasis-related gene
expression in metastatic human oral carcinoma
sublines selected from a poorly metastatic parental
cell line. Cancer. 2002;95(8):1663–1672.
18. Xing J, Ginty DD, Greenberg ME. Coupling of the
RAS-MAPK pathway to gene activation by RSK2,
a growth factor-regulated CREB kinase. Science.
19. Jean D, Bar-Eli M. Regulation of tumor growth
and metastasis of human melanoma by the CREB
transcription factor family. Mol Cell Biochem. 2000;
20. Jean D, et al. Inhibition of tumor growth and metas-
tasis of human melanoma by intracellular anti-
ATF-1 single chain Fv fragment. Oncogene. 2000;
21. Jean D, Harbison M, McConkey DJ, Ronai Z, Bar-
Eli M. CREB and its associated proteins act as sur-
vival factors for human melanoma cells. J Biol Chem.
22. Xie S, Price JE, Luca M, Jean D, Ronai Z, Bar-Eli M.
Dominant-negative CREB inhibits tumor growth
and metastasis of human melanoma cells. Onco-
23. Miron T, Vancompernolle K, Vandekerckhove J,
Wilchek M, Geiger B. A 25-kD inhibitor of actin
polymerization is a low molecular mass heat shock
protein. J Cell Biol. 1991;114(2):255–261.
24. Zhang D, Wong LL, Koay ES. Phosphorylation of
Ser78 of Hsp27 correlated with HER-2/neu status
and lymph node positivity in breast cancer. Mol
25. Shin KD, et al. Blocking tumor cell migration and
invasion with biphenyl isoxazole derivative KRIBB3,
a synthetic molecule that inhibits Hsp27 phosphor-
ylation. J Biol Chem. 2005;280(50):41439–41448.
26. Gerthoffer WT, Gunst SJ. Invited review: focal
adhesion and small heat shock proteins in the
regulation of actin remodeling and contractility in
smooth muscle. J Appl Physiol. 2001;91(2):963–972.
27. Landry J, Huot J. Modulation of actin dynamics
during stress and physiological stimulation by
a signaling pathway involving p38 MAP kinase
and heat-shock protein 27. Biochem Cell Biol. 1995;
28. Gabarra-Niecko V, Schaller MD, Dunty JM. FAK
regulates biological processes important for the
pathogenesis of cancer. Cancer Metastasis Rev. 2003;
29. Mon NN, Ito S, Senga T, Hamaguchi M. FAK sig-
naling in neoplastic disorders: a linkage between
inflammation and cancer. Ann N Y Acad Sci. 2006;
30. Maina F, et al. Uncoupling of Grb2 from the Met
receptor in vivo reveals complex roles in muscle
development. Cell. 1996;87(3):531–542.
31. Bardelli A, et al. Uncoupling signal transducers
from oncogenic MET mutants abrogates cell trans-
formation and inhibits invasive growth. Proc Natl
Acad Sci U S A. 1998;95(24):14379–14383.
32. Takayama H, et al. Diverse tumorigenesis associ-
ated with aberrant development in mice overex-
pressing hepatocyte growth factor/scatter factor.
Proc Natl Acad Sci U S A. 1997;94(2):701–706.
33. Rong S, Segal S, Anver M, Resau JH, Vande Woude
GF. Invasiveness and metastasis of NIH 3T3 cells
induced by Met-hepatocyte growth factor/scatter
factor autocrine stimulation. Proc Natl Acad Sci U S A.
34. Wu D, et al. cAMP-responsive element-binding pro-
tein regulates vascular endothelial growth factor
expression: implication in human prostate cancer
bone metastasis. Oncogene. 2007;26(35):5070–5077.
35. Benjamin IJ, McMillan DR. Stress (heat shock)
proteins: molecular chaperones in cardiovascular
biology and disease. Circ Res. 1998;83(2):117–132.
36. Arrigo AP, et al. Hsp27 (HspB1) and alphaB-crys-
tallin (HspB5) as therapeutic targets. FEBS Lett.
37. Garrido C, Brunet M, Didelot C, Zermati Y, Schmitt
E, Kroemer G. Heat shock proteins 27 and 70: anti-
apoptotic proteins with tumorigenic properties.
Cell Cycle. 2006;5(22):2592–2601.
38. Lavoie JN, Lambert H, Hickey E, Weber LA, Landry J.
Modulation of cellular thermoresistance and actin
filament stability accompanies phosphorylation-
induced changes in the oligomeric structure of heat
shock protein 27. Mol Cell Biol. 1995;15(1):505–516.
39. Geum D, Son GH, Kim K. Phosphorylation-depen-
dent cellular localization and thermoprotective role
of heat shock protein 25 in hippocampal progeni-
tor cells. J Biol Chem. 2002;277(22):19913–19921.
40. Kang S, et al. FGFR3 activates RSK2 to mediate
hematopoietic transformation through tyrosine
phosphorylation of RSK2 and activation of the MEK/
ERK pathway. Cancer Cell. 2007;12(3):201–214.
41. Gibson SL, Ma Z, Shaw LM. Divergent roles for
IRS-1 and IRS-2 in breast cancer metastasis. Cell
42. Ma Z, Gibson SL, Byrne MA, Zhang J, White MF,
Shaw LM. Suppression of insulin receptor sub-
strate 1 (IRS-1) promotes mammary tumor metas-
tasis. Mol Cell Biol. 2006;26(24):9338–9351.
43. Zhang Y, et al. c-Jun, a crucial molecule in metas-
tasis of breast cancer and potential target for bio-
therapy. Oncol Rep. 2007;18(5):1207–1212.
44. Zhang Y, et al. Critical role of c-Jun overexpression
in liver metastasis of human breast cancer xeno-
graft model. BMC Cancer. 2007;7:145.
45. Robinson CM, et al. Overexpression of JunB in
undifferentiated malignant rat oral keratinocytes
enhances the malignant phenotype in vitro without
altering cellular differentiation. Int J Cancer. 2001;
46. Benvenuti S, Comoglio PM. The MET receptor
tyrosine kinase in invasion and metastasis. J Cell
47. Furge KA, Zhang YW, Vande Woude GF. Met recep-
tor tyrosine kinase: enhanced signaling through
adapter proteins. Oncogene. 2000;19(49):5582–5589.
48. Ma PC, Maulik G, Christensen J, Salgia R. c-Met:
structure, functions and potential for therapeutic
inhibition. Cancer Metastasis Rev. 2003;22(4):309–325.
49. Baldassarre G, et al. p27(Kip1)-stathmin interac-
tion influences sarcoma cell migration and inva-
sion. Cancer Cell. 2005;7(1):51–63.
50. Platet N, Cathiard AM, Gleizes M, Garcia M. Estro-
gens and their receptors in breast cancer progres-
sion: a dual role in cancer proliferation and inva-
sion. Crit Rev Oncol Hematol. 2004;51(1):55–67.
51. Chen AG, Yu ZC, Yu XF, Cao WF, Ding F, Liu ZH.
Overexpression of Ets-like protein 1 in human
esophageal squamous cell carcinoma. World J Gas-