Blimp1 Activation by AP-1 in Human Lung Cancer Cells
Promotes a Migratory Phenotype and Is Inhibited by the
Lysyl Oxidase Propeptide
Ziyang Yu1., Seiichi Sato1., Philip C. Trackman2, Kathrin H. Kirsch3, Gail E. Sonenshein1*
1Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 2Division of Oral Biology, Boston University Henry M.
Goldman School of Dental Medicine, Boston, Massachusetts, United States of America, 3Department of Biochemistry, Boston University School of Medicine, Boston,
Massachusetts, United States of America
B lymphocyte-induced maturation protein 1 (Blimp1) is a master regulator of B cell differentiation, and controls migration of
primordial germ cells. Recently we observed aberrant Blimp1 expression in breast cancer cells resulting from an NF-kB RelB
to Ras signaling pathway. In order to address the question of whether the unexpected expression of Blimp1 is seen in other
epithelial-derived tumors, we selected lung cancers as they are frequently driven by Ras signaling. Blimp1 was detected in
all five lung cancer cell lines examined and shown to promote lung cancer cell migration and invasion. Interrogation of
microarray datasets demonstrated elevated BLIMP1 RNA expression in lung adenocarcinoma, pancreatic ductal carcinomas,
head and neck tumors as well as in glioblastomas. Involvement of Ras and its downstream kinase c-Raf was confirmed using
mutant and siRNA strategies. We next addressed the issue of mechanism of Blimp1 activation in lung cancer. Using
knockdown and ectopic expression, the role of the Activator Protein (AP)-1 family of transcription factors was demonstrated.
Further, chromatin immunoprecipitation assays confirmed binding to identified AP-1 elements in the BLIMP1 promoter of
ectopically expressed c-Jun and of endogenous AP-1 subunits following serum stimulation. The propeptide domain of lysyl
oxidase (LOX-PP) was identified as a tumor suppressor, with ability to reduce Ras signaling in lung cancer cells. LOX-PP
reduced expression of Blimp1 by binding to c-Raf and inhibiting activation of AP-1, thereby attenuating the migratory
phenotype of lung cancer cells. Thus, Blimp1 is a mediator of Ras/Raf/AP-1 signaling that promotes cell migration, and is
repressed by LOX-PP in lung cancer.
Citation: Yu Z, Sato S, Trackman PC, Kirsch KH, Sonenshein GE (2012) Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype
and Is Inhibited by the Lysyl Oxidase Propeptide. PLoS ONE 7(3): e33287. doi:10.1371/journal.pone.0033287
Editor: Vladimir V. Kalinichenko, Cincinnati Children’s Hospital Medical Center, United States of America
Received October 26, 2011; Accepted February 10, 2012; Published March 15, 2012
Copyright: ? 2012 Yu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were supported by National Institutes of Health (NIH) grants R01 CA143108 and PO1 ES011624. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
B lymphocyte-induced maturation protein 1 (Blimp1) or
Positive-Regulatory Domain I Binding Factor 1 (PRDI-BF1) is a
zinc finger protein encoded by the PRDI-BF1 and RIZ domain 1
(PRDM1) or BLIMP1 gene [1,2], which was initially isolated as a
transcriptional repressor of the IFNb promoter . Several
mechanisms of Blimp1-mediated repression of gene transcription
have been elucidated: recruitment of histone methyltransferases
(HMTs) , histone deacetylases (HDACs)  or corepressors 
or by competition with transcriptional activators . Blimp1 was
identified as a master regulator of B cell terminal differentiation
, which promotes differentiation of B lymphocytes to plasma
cells . Several factors have been implicated in the activation of
transcription of the Blimp1 gene during the differentiation of B
cells, including NF-kB, AP-1, IRF4, STAT3 and STAT5,
although, their precise mechanisms of action are not fully
understood . Blimp1 was subsequently shown to regulate T
cell proliferation and homeostasis . During development,
Blimp1 controls primordial germ cell (PGC) specification and
migration as Blimp1-deficient mouse embryos generate PGC-like
cells which fail to show characteristic PGC migration [11,12].
Somewhat unexpectedly, Blimp1 was detected in non-hematopoi-
etic cancer cells. Our laboratory observed Blimp1 expression in
breast cancer cells, and showed it repressed transcription of the
ESR1 gene encoding estrogen receptor alpha (ERa), thereby
promoting a more migratory phenotype . Transcriptional
induction of Bcl-2 levels by the NF-kB RelB subunit recruited Ras
to the mitochondria . The resultant Ras signaling led to an
aberrant induction of Blimp1 in the breast cancer cells . The
exact transcription factor(s) downstream of Ras that mediated the
activation of Blimp1 in these cancer cells remained to be
identified. However, the involvement of Ras signaling in Blimp1
activation leads us to hypothesize that expression of Blimp1 may
be more widespread in cancer than previously realized. Colorectal
tumor cells were also found to express Blimp1, which repressed the
TP53 gene and thus maintained cell growth .
Lung cancer is the leading cause of cancer-related death in
Western countries. Approximately two-thirds of patients are
diagnosed at an advanced stage, and of the remaining patients
who undergo surgery, 30–50% develop recurrence with metastatic
disease [16,17]. The RAS oncogene is mutated in up to ,30% of
PLoS ONE | www.plosone.org1March 2012 | Volume 7 | Issue 3 | e33287
lung cancers, with the majority of mutations found in the KRAS
gene [16,17]. Oncogenic K-Ras predisposes transgenic mice to
lung tumorigenesis . Ras signals via multiple pathways,
including mitogen activated protein kinase (MAPK). As nuclear
acceptors for MAPK signaling cascades, the activator protein (AP)-
1 family of transcription factors has been implicated in the highly
migratory phenotype of lung cancer cells [19,20,21].
The lysyl oxidase (LOX) gene was isolated as the ras recision gene (rrg)
due to its ability to revert Ras-mediated transformation of NIH
3T3 fibroblasts . Our group showed ectopic Pro-LOX
expression reduced extracellular signal-regulated kinase (ERK)
and phosphatidylinositol 3-kinase (PI3K)/Akt signaling and
activation of NF-kB in Ras-transformed NIH 3T3 cells . Loss
of LOX gene expression was seen in many cancerous tissues and
derived cell lines including those from lung [24,25,26], colon ,
prostate , gastric  and head and neck squamous cancers
. Ectopic LOX gene expression reduced colony formation of
cultured gastric cancer cells and tumor formation in a xenograft
model . Lysyl oxidase is synthesized and secreted as a pro-
enzyme (Pro-LOX), and processed to a functional enzyme (LOX)
and amino terminal propeptide (LOX-PP) . The rrg activity of
Pro-LOX was unexpectedly mapped to the LOX-PP domain, as
judged by inhibition of the transformed phenotype of NIH 3T3-
Ras cells . Subsequently, LOX-PP was shown to reduce the
migratory phenotype of mouse breast cancer cells driven by Her-
2/Neu, which signals via Ras and their ability to form tumors in a
nude mouse xenograft model [33,34]. In H1299 lung cancer cells,
which contain a mutant NRAS gene, LOX-PP reduced the
activation of ERK and Akt, and ability for anchorage-independent
growth and invasive colony formation in Matrigel . LOX-PP
also attenuated fibronectin-mediated activation of focal adhesion
kinase in breast cancer cells [34,35], and fibroblast growth factor
(FGF)-2-induced proliferation of prostate cancer cells . Here
we asked whether Blimp1 is expressed in lung cancer cells given
the important role of Ras signaling in these cancer cells. Blimp1
was detected in all lung cancer lines examined and promoted their
migration and invasion. Furthermore, BLIMP1 RNA was detected
in other primary tumors driven by Ras signaling. In lung cancer
cells, Blimp 1 expression was induced by a Ras/c-Raf/AP-1
pathway, which could be inhibited by LOX-PP via interaction
with c-Raf. Thus, these studies identify Blimp1 as a critical
mediator of lung cancer cell migratory phenotype by the
transforming Ras/c-Raf/AP-1 cascade.
Materials and Methods
Cells and culture conditions
The non-small cell lung cancer (NSCLC) A549 and H1299 cell
lines were kindly provided by Zhi-Xiong Jim Xiao (Boston
University School of Medicine, Boston MA). The Calu-1, H23 and
H441 cell lines were generously provided by Hasmeena Kathuria
and Maria Ramirez (Boston University School of Medicine).
A549, Calu-1, H23 and H441 cells express mutant K-Ras [37,38]
and H1299 express mutant N-Ras . Bosc23 cells were
obtained from the American Type Culture Collection (ATCC).
All cell lines were maintained in Dulbecco’s Minimal Essential
Medium except H441 which was maintained in RPMI-1640. The
culture media were supplemented with 10% fetal bovine serum
(FBS), as recommended by the ATCC. H1299 clones expressing
mouse LOX-PP in a doxycycline (dox) inducible vector were
established and total RNA isolated as described previously .
Inducible stable A549 cells expressing V5-tagged human or mouse
LOX-PP were established as previously described [33,34]. Briefly,
pCL-Ampho retrovirus packaging vector (Imgenex, San Diego,
CA) was co-transfected into BOSC 23 cells using FuGENE 6
(Roche Diagnostics Co., Indianapolis, IN) with either empty
effector vector pC4bsrR(TO) (EV) or vector bearing the DNA
fragments of human or mouse LOX-PP with C-terminal V5 tag
and the regulator vector pCXneoTR2 (both kindly provided by
Tsuyoshi Akagi, KAN, Kobe, Japan). After 48 h, supernatants
containing viral particles were harvested and passed through a
0.45 mm filter (Corning Inc., Corning, NY). A549 lung cancer cells
were dually infected for 48 h with supernatant from BOSC 23
cells containing viruses that carry the regulator and effector vectors
supplemented with 6 mg/ml polybrene (Sigma, St. Louis, MO).
Infected cells were selected with 10 mg/ml blasticidin (Invitrogen,
Carlsbad, CA) and 1.4 mg/ml geneticin (Sigma) to generate
separate pools of stable A549-EV, A549-human LOX-PP and
A549-mouse LOX-PP cells.
Plasmids and transfection analysis
The pcDNA3/Blimp  and the 7-kB Blimp1-pGL3 luciferase
reporter (Blimp1-luc)  vectors were kindly provided by Tom
Maniatis (Columbia University, NY) and Kathryn Calame
(Columbia University), respectively. The c-Jun, c-Fos, Fra-1 and
Fra-2 AP-1 constructs in pCI expression vector were as previously
reported . For transient transfection of expression vectors,
cultures in 12-well plates were incubated for 48 h in the presence
of 1 mg DNA and 3 ml Fugene 6 or 2.5 ml Lipofectamine 2000
(Invitrogen). Co-transfection of the MSV-b-gal vector, expressing
b-galactosidase (b-gal) was used to normalize for transfection
efficiency. All transient transfection reporter assays were per-
formed, in triplicate, two times as described previously , and
the standard error of the mean (SEM) calculated. BLIMP1 siRNA,
and JUN, FRA-1 and FRA-2 siRNA duplex sequences were as
previously described [15,43]. The siRNA targeting human KRAS
gene (sc-35731) was from Santa Cruz Biotechnology (Santa Cruz,
CA). The RNA duplexes used for targeting c-RAF were as
described by Chadee and Kyriakis  and purchased from
QIAGEN (Valencia, CA). For transient transfection of single
siRNAs, cultures in 6-well plates were incubated for 24 h in the
presence of siRNA duplex (10 nM final) and Lipofectamine
RNAiMax (Invitrogen), according to the manufacturer’s protocol.
In the case of co-transfection of two AP-1 siRNAs, the final
concentration of each siRNA was 10 nM, making the total siRNA
concentration 20 nM. Where mentioned, the culture was
supplemented with a negative control siRNA (Qiagen) at a final
concentration of either 10 or 20 nM, as appropriate. The Ras
S186 expression vector was kindly provided by Mark Philips (NYU
School of Medicine, New York, NY). For construction of N-
terminally glutathione S-transferase (GST) tagged LOX-PP and its
deletion mutants, the cDNA encoding full length LOX-PP (WT,
amino acid 1–162) and deletion of aa residues 26–100 (DM3) were
amplified from full-length Pro-LOX cDNA  and inserted into
the BamHI/ClaI site of pEBG-GST mammalian expression
vector, a generous gift of Dr. Bruce Mayer (University of
Connecticut Health Center, Farmington, CT). For construction
of C-terminally GST-tagged LOX-PP, the cDNAs encoding GST
and LOX-PP were amplified and inserted into pcDNA3.1 (+).
pBabe-puro-MEK1 S217E/S221E constitutively active (CA-
MEK) mutant was kindly supplied by Dr. Geoffrey M. Cooper
(Boston University, Boston, MA). The cDNA encoding MEK1
S217E/S221E was inserted into pcDNA3.1(+).
Nuclear extracts (NE) and whole cell extracts (WCE) were
prepared and subjected to immunoblotting, as described previ-
ously . For the detection of secreted recombinant LOX-PP-
Ras/c-Raf to AP-1 Pathway Activates Blimp1
PLoS ONE | www.plosone.org2March 2012 | Volume 7 | Issue 3 | e33287
V5, culture medium (40 ml from 2 ml of culture medium) was
immunoblotted using an anti-V5 antibody (R960-25, Invitrogen).
The antibodies against Blimp1 (no. 9115s), c-Jun (no. 9165),
phospho-c-Jun (no. 9261s), MEK1/2 (L38C12; no. 4694) phos-
pho-ERK1/2 (phospho-Thr202/Tyr204; no. 9101s) and ERK1/
2 (9102) were obtained from Cell Signaling (Danvers, MA).
Antibodies against GST (B-14), K-Ras (F234), B-Raf (F-7), Fra-1
(N-17), Fra-2 (Q-20) and c-Fos (H-125) were from Santa Cruz
Biotechnology. Antibodies against b-actin (AC-15) and a-tubulin
(DM1A) were from Sigma. Hsp70/Hsc70 (SPA-820) and Hsp90
(SPA-830) antibodies were purchased from Stressgen (Victoria,
BC, Canada). Antibody against c-Raf (clone 53) and Ras (clone
18/Ras) were from BD Transduction (Franklin Lakes, NJ). Rabbit
polyclonal antibodies against LOX-PP were prepared as described
previously . The results from a minimum of two independent
experiments were subjected to densitometry and normalized to a
b-actin loading control and the mean values relative to control
empty vector (EV) cells (set to 1.0) given.
Migration and invasion assays
Suspensions of 16105cells were layered, in triplicate, in the
upper compartments of Costar Transwells (Corning, Lowell, MA)
on an 8-mm diameter polycarbonate filter (8 mm pore size), and
incubated at 37uC for 16 h. Migration of the cells to the lower side
of the filter was evaluated with the phosphatase enzymatic assay
using p-nitrophenyl phosphate and OD410 nmdetermination, as
described previously  or by staining with crystal violet and
OD570 nmdetermination (63). The average migration from three
independent experiments 6 SD is presented relative to the control
EV, which was set at 1.0. P values were calculated using a
Student’s t-test. For invasion assays, filters were precoated with
10 mg of Matrigel (BD Biosciences, San Jose, CA). Migration of
the cells to the lower side of the filter was evaluated by staining
with crystal violet and OD570 nmdetermination. The mean 6 SD
are presented. Invasion assays were performed three times, in
Reverse Transcriptase (RT)-PCR analysis
RNA was isolated using RNeasy Mini Kit (Invitrogen), and
samples with A260/A280ratios between 1.8 and 2.0 were treated
with RQ1 RNase-free DNase (Promega). Superscript III RT was
used for reverse transcription with 1 mg RNA in the presence of
100 ng of random primers (Invitrogen). For Realtime quantitative
PCR (Q-PCR), the BLIMP1 primers were as described previously
. The GAPDH primers were: Forward 59-TTGCCATCAAT-
GACCCCTTCA-39; Reverse 59-CGCCCCACTTGATTTTG-
GA-39. Q-PCR was performed in triplicate in a Roche LightCycler
Chromatin Immunoprecipitation (ChIP) assay
ChIP assays were performed using an EZ-ChIP kit (Millipore
Corporation, Billerica, MA), according to the manufacturer’s
instructions. For analysis of ectopically expressed AP-1, 24 h after
H441 cells were transfected with a c-Jun expression vector,
formaldehyde (1% final) was added to the cell culture medium.
Whole cell lysates were made and subjected to sonication in a
Misonix 3000 Sonicator (Misnonix, Farmingdale, NY) for 15 cycles
of10 seceachtoyieldgenomicDNAfragmentsof,200to1000 bp.
After preclearing with ChIP grade Protein G agarose, 100 ml of
sheared DNA-protein complexes were immunoprecipitated with
antibodies against c-Jun (sc-1694) or normal rabbit IgG (sc-2027)
(Santa Cruz Biotechnology). Crosslinking was reversed and purified
genomic DNA fragments were subjected to PCR. The crosslinking
was reversed by overnight incubation at 65uC and genomic DNA
fragments purified with a Qiaquick PCR purification kit (QIAGEN,
no. 28104). Two binding elements for AP-1, which are also known
as TPA responsive elements or TREs, were previously identified at
21813 and 21647 bp relative to the BLIMP1 transcription start site
and verifiedusing TransFac(genomatix.de)analysis.Theregion
across the two TREs was amplified by PCR. The primers for the
21813 bp TRE: Forward 59-GCCTTCTTCCCACCTCAAA-
TATCA-39, Reverse 59-TGGCCTGCTGTTCAAACAGTCT-
CA-39; and for the 21647 bp TRE: Forward 59-GTTGCAT-
GATGGTGTATGTGGCCT-39, Reverse 59-ATCCAGCCTG-
CTCAAGAGGGTTTA -39. As a positive control for AP-1 binding,
a fragment of the human JUN promoter containing two closely
analysis, using the previously described primers . As a negative
control, primers were designed for an upstream region of the
BLIMP1 promoter (25508 to 25366 bp) containing no TRE sites:
Forward 59- TCCTTCCCTGTGTTTGGTCCCATT-39, Reverse
59-ATTGTTTCCTTCAAGCAGGCACCC-39. For binding of
endogenous AP-1 subunits, A549 cells were incubated in serum-
free medium for 48 h and FBS (10% final concentration) added
back. After 30 min, WCE were prepared and subjected to ChIP
assay, as above, using antibodies against normal rabbit IgG, c-Jun,
Fra-1 (sc-183), or Fra-2 (sc-604) (from Santa Cruz Biotechnology).
Immunoprecipitation and GST pull down assay
H1299 or A549 cells were lysed with Buffer A [25 mM HEPES-
KOH (pH 7.2), 150 mM KCl, 2 mM EDTA, 1 mM phenyl-
methylsulfonyl fluoride, 1 mM dithiothreitol, 0.5 mg/ml leupeptin,
2 mM pepstatin A, 1 mg/ml aprotinin, and 1% Triton X-100].
The lysates were centrifuged in a microcentrifuge for 10 min at
13,000 rpm at 4uC to remove insoluble material. For immuno-
precipitation, 2 mg of either rabbit anti-LOX-PP  or rabbit
control IgG was added to 500 mg cell lysate, followed by overnight
incubation at 4uC. Protein G-Sepharose beads (Invitrogen) were
then added to the mixture, followed by incubation at 4uC for 2 h
with gentle shaking. The beads were washed four times with Buffer
A. For GST-pull down assay, the lysates were incubated with 20 ml
Glutathione-Sepharose 4B (GE Healthcare) for 2 h at 4uC. The
resin was washed four times with Buffer A. The immune-
complexes or GST pull down-complexes were eluted from the
Sepharose beads with SDS-PAGE sample buffer, and the
precipitated proteins analyzed by immunoblot analysis.
Lung cancer cells express Blimp1
Five lung cancer cell lines, driven by mutant K-Ras or N-Ras,
were selected to test for Blimp1 expression: A549, H1299, Calu-1,
H23 and H441. Nuclear extracts were subjected to immunoblot
analysis (Fig. 1A). As a reference, we included nuclear extracts
from ERa positive MCF-7 and ERa negative MDA-MB-231
breast cancer cells, which displayed relatively lower and higher
Blimp1 levels, respectively . All five lung cancer cell lines
expressed 100 kDa Blimp1 protein recognized by an antibody
against the N-terminus of the human Blimp1 protein. As seen
previously, the ERa negative MDA-MB-231 breast cancer cells
expressed higher levels of Blimp1 than the ERa positive MCF-7
cells . All of the lung cancer cells expressed substantially higher
amounts of Blimp1 than the MDA-MB-231 line. Thus, Blimp1 is
expressed in lung cancer cells.
Blimp1 promotes migration of lung cancer cells
The absence of Blimp1 in mouse embryos led to development of
primordial germ-like cells that were unable to migrate [11,12]. To
Ras/c-Raf to AP-1 Pathway Activates Blimp1
PLoS ONE | www.plosone.org3March 2012 | Volume 7 | Issue 3 | e33287
test whether Blimp1 expression is involved in control of lung
cancer cell migration, a knockdown strategy was used. A549 and
H1299 cells, which displayed relatively high levels of Blimp1
(Fig. 1A), were incubated with either siBLIMP1-1 or siBLIMP1-2,
two independent siRNA species, or with a scrambled negative
control siRNA. After 48 h, samples of WCE were subjected to
immunoblot analysis. Both BLIMP1 siRNAs resulted in effective
knockdown of Blimp1 protein expression compared to the control
siRNA. A more robust knockdown was seen with siBLIMP1-2 in
both cell lines, i.e., 93% decrease in A549 and 88% in H1299
compared to 30% in A549 and 48% in H1299 with siBLIMP1-1
(upper panels, Figs. 1B and 1C). The effects of a 24 h incubation
with these siRNAs on migration of A549 and H1299 cells were
tested in Boyden chambers (16105per well) using FBS as the
chemo-attractant. Cell migration was measured 16 h later.
Knockdown of BLIMP1 expression led to decreased migration of
A549 (Fig. 1B) and H1299 (Fig. 1C) lung cancer cells. In three
independent experiments, performed in triplicate, siBLIMP1-2 led
to a more profound reduction in migration of A549 (average
decreases of 42% with siBLIMP1-1 vs 71% with siBLIMP1-2) and
H1299 cells (average decreases of 35% with siBLIMP1-1 vs 54%
with siBLIMP1-2). No significant effects of the treatments on cell
proliferation were observed (data not shown). These results are
consistent with the reduction of Blimp-1 levels. To confirm that
the reduction of cell migration was specifically due to the
knockdown of Blimp1 expression, we performed a rescue
experiment using siBLIMP1-2 and Blimp1 ectopic expression in
A549 lung cancer cells. Briefly, A549 cells were incubated with
either siBLIMP1-2 or negative control siRNA for 16 h followed by
transient transfection of a vector expressing Blimp1 or EV DNA.
After 32 h, cells were subjected to a migration assay or samples of
WCE were subjected to immunoblot analysis. BLIMP1 siRNA-2
resulted in effective knockdown of Blimp1 protein expression
compared to the control siRNA and this effect was overcome by
the Blimp1 expression vector (Fig. 1D, inset). As seen above,
knockdown of BLIMP1 expression led to a 42% decrease in cell
migration compared to cells transfected with negative control
siRNA and EV, and this was overridden by ectopic Blimp1
expression (Fig. 1D). A 33% increase in migration of A549 cells
transfected with Blimp1 cDNA and siBlimp1-2 was observed
compared to the control siRNA and EV transfected cells. In
addition, no significant effects on cell proliferation were noted over
Figure 1. Blimp1 is expressed in lung cancer cells and its
knockdown reduces migration. (A) Samples of nuclear extracts
(20 mg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells
and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were
subjected to immunoblotting for Blimp1 and b-actin, as a control for
equal loading. Positions of molecular weight markers are given in the
left lane. A representative of two independent experiments with similar
results is shown. (B) A549 and (C) H1299 cells were transiently
transfected with 10 nM each of siBLIMP1-1, siBLIMP1-2 or a scrambled
negative control siRNA. Upper panels: Forty-eight h after transfection,
WCE (30 mg) were subjected to immunoblotting for Blimp1 and b-actin.
The bands were quantified using NIH Image J software and Blimp1
expression normalized to b-actin expression. Normalized Blimp1
expression was determined in two independent experiments and the
average values are given below the blots. Lower panels: Alternatively,
after 24 h, cultures were trypsinized and 16105cells subjected to a
migration assay for 16 h, in triplicate. The average migration from three
independent experiments 6 SD is presented relative to the negative
control siRNA (set at 1.0). P values were calculated using Student’s t-
test. *, P,0.005; **, P,0.0005. (D) A549 cells were incubated in the
presence of 0.5 nM siBlimp1-2 or scrambled negative control siRNA for
16 h. Cells were then transfected with Blimp1 expression vector (2 mg
per well in 6-well plate) and incubated for 32 h. (Inset) Whole cell
lysates (20 mg) were subjected to western blot analysis using antibodies
against Blimp1 or b-actin. Cultures were trypsinized and 16105cells
subjected to a migration assay for 16 h, in triplicate. The average
migration from two independent experiments 6 SE is presented
relative to the negative control siRNA and EV (set at 1.0). Data shown is
a representative of two independent experiments with similar results.
(E) A549 cells were transiently transfected with 10 nM each of siBLIMP1-
1, siBLIMP1-2 or a scrambled negative control siRNA. After 48 h, cultures
were trypsinized and 16105cells subjected to an invasion assay for
16 h, in triplicate. The average data from three independent
experiments 6 SD is presented relative to the negative control siRNA
(set at 1.0). P values were calculated using Student’s t-test. *, P,0.01.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
PLoS ONE | www.plosone.org4March 2012 | Volume 7 | Issue 3 | e33287
the time course (data not shown). Next, we tested the effects of
Blimp1 knockdown on invasion. A decrease in invasion by A549
lung cancer cells was noted with BLIMP1 siRNA-1, which was
even more profound with BLIMP1 siRNA-2 compared to the
negative control siRNA, consistent with the migration data
(Fig. 1E). Thus, reduced levels of Blimp1 lead to decreased ability
of A549 and H1299 lung cancer cells to migrate and invade.
We next performed the converse experiment and ectopically
expressed Blimp1 in A549 and H441 cells, which express higher
and moderate levels of Blimp1, respectively. Cultures were
transiently transfected with a Blimp1 expression cDNA or parental
empty vector (EV) for 24 h and subjected to migration assays, as
above. In three independent experiments, performed in triplicate,
Blimp1 overexpression increased migration of A549 and H441
cells by an average of 64% (Fig. 2A) and 58% (Fig. 2B),
respectively. Western blotting of extracts prepared from similarly
transfected cultures confirmed ectopic expression of Blimp1 (upper
panels, Figs. 2A and 2B). Thus, Blimp1 promotes a more
migratory phenotype of lung cancer cells.
Multiple primary tumors display overexpression of
We next asked whether BLIMP1 RNA is detected in primary
lung tumors. Elevated BLIMP1 mRNA expression was detected
in lung adenocarcinoma samples compared to normal lung tissues
 (Fig. 2C). Constitutive Ras signaling induced by either a
mutant RAS gene or upstream activator such as growth factor
receptor has been implicated in many other tumors. KRAS
mutations have been found in .95% of pancreatic ductal
adenocarcinomas , while overexpression of Epidermal
Growth Factor receptor (EGFR), which induces Ras signaling,
was found in 80–90% human head and neck squamous cell
carcinomas  and 40% of glioblastomas . Notably, our
analyses using microarray datasets in Oncomine revealed
elevated BLIMP1 RNA expression in samples of pancreatic
adenocarcinoma , tongue squamous cell carcinoma  and
glioblastoma  compared to the corresponding normal tissues
(Fig. 2D). Thus, BLIMP1 RNA is overexpressed in a diverse
group of human cancers.
Figure 2. Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells
were transiently transfected with 1 mg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and
subjected to immunoblot analysis for Blimp1 and b-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration
assay as in Fig. 1. The average migration from three independent experiments 6 SD is presented relative to the EV (set at 1.0). P values were
calculated using a Student’s t-test. *, P,0.005. C) Box plot from the Hou lung cancer microarray dataset was accessed using Oncomine Database.
Student’s t-test for the two groups shows a P value of 0.024. D) Box plots from the Badea pancreatic cancer, Estilo head-neck cancer and Sun brain
tumor microarray datasets were accessed using Oncomine Database. Student’s t-tests comparing the groups in these studies show P values of
8.67e27, 0.001 and 3.28e215, respectively.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
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Ras to c-Raf signaling induces Blimp1 expression in lung
To directly address the role of Ras signaling on Blimp1 levels in
lung cancer cells, a dominant negative mutant was first used. The
Ras S186 mutant retains the ability to associate with the effector
protein kinase c-Raf but does not translocate to the membrane and
inhibited activation of Blimp1 by Bcl-2 [13,56]. A549 cells, which
express an activated mutant K-Ras C12, were transfected with EV
or a plasmid expressing Ras S186 and after 48 h, WCE and RNA
were isolated. Ectopic expression of Ras S186, which was
confirmed by immunoblotting, decreased Blimp1 protein expres-
sion by ,54% (Fig. 3A). In two separate experiments, BLIMP1
mRNA expression declined an average of 48% upon ectopic
expression of Ras S186 (Fig. 3B). The effects of the dominant
Figure 3. A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 mg of a plasmid
expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 mg) of WCE were subjected to immunoblot
analysis for Blimp1, Ras and a-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to b-actin
expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was
isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH. The values represent an
average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 mg of Ras S186 plasmid or EV DNA, 0.33 mg of a MSV-
b-gal expression vector and 0.16 mg of the 7-kB Blimp1 promoter Blimp1-Luc, in a 12-well plate. After 48 h, cell lysates were subjected to
measurements for luciferase and b-gal activities and normalized Blimp1 promoter activity values are presented as the mean 6 SEM from two
experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of
25 ml of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.46105) were seeded at a final siRNA concentration of 20 nM for
48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and a-tubulin. Average normalized levels of
Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two
independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c-RAF or a negative control siRNA was
incubated in the presence of 25 ml of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.46105) were seeded at a final siRNA
concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and a-tubulin. Average normalized
levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one
of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative
control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1-luc promoter construct (0.16 mg) and an MSV- b-gal expression
vector (0.33 mg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are
presented as the mean 6 SEM from two experiments (EV DNA set to 1.0).
Ras/c-Raf to AP-1 Pathway Activates Blimp1
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negative Ras on Blimp1 promoter activity were also tested. A549
cells were co-transfected with either EV or Ras S186 vector DNA,
along with a 7-kB Blimp1 promoter reporter construct Blimp1-luc
and a b-gal expression vector, for normalization of transfection
efficiencies. Overexpression of Ras S186 led to an average
decrease of 69% in normalized Blimp1 promoter activity
(Fig. 3C). Lastly, an si-KRAS strategy was employed (Fig. 3D).
Knockdown of K-Ras led to a decrease of 93% of K-Ras protein
expression and to a substantial decrease in ERK activity as judged
by a reduction in phospho-ERK levels. Furthermore, an average
decrease of 44% in Blimp1 levels were seen in two independent
experiments (Fig. 3D). Together, these results indicate that
oncogenic Ras signaling in A549 lung cancer cells drives BLIMP1
Ras mediates its effects by signaling via several pathways. The c-
Raf/Erk pathway has been implicated in control of migration and
thus we used a knockdown strategy to test whether c-Raf mediates
signals leading to induction of Blimp1. A549 cells were transfected
for 48 h with a negative control siRNA or an siRNA against c-
RAF, which effectively decreased levels of c-Raf and Blimp1
protein, which decreased by an average of 39% in two
experiments (Fig. 3E). To confirm a role of c-Raf in Blimp1
promoter activity, A549 cells were reverse-transfected with c-RAF
siRNA or control siRNA, and after 8 h were transfected with a
Blimp1 reporter construct for 40 h. The si-c-RAF led to an average
decrease of 52% in Blimp1 promoter activity compared to negative
control siRNA (Fig. 3F). Thus, a Ras to c-Raf pathway activates
BLIMP1 gene expression in A549 lung cancer cells.
AP-1 induces Blimp1 expression
The AP-1 family of transcription factors has been implicated in
the highly migratory phenotype of lung cancer cells [19,20,21],
and two functional AP-1 binding sites or TREs have been
identified in the BLIMP1 promoter . Substantial decreases in
amounts of c-Jun (53%), Fra-1 (65%) and Fra-2 (43%) resulted
from treatment with the c-RAF siRNA (Fig. 3E), consistent with
the observed reduction in Blimp1 expression. Similarly knock-
down of Ras led to average decreases of 35, 33 and 28% in levels
of c-Jun, Fra-1 and Fra-2, respectively (Fig. 3D). We next
characterized AP-1 subunit expression in the lung cancer cell
lines by subjecting nuclear extracts to immunoblot analysis
(Fig. 4A). High levels of c-Jun were detected in H1299 and
Calu-1 cells and low to moderate c-Jun levels in H23, H441 and
A549 cells. High levels of Fra-1 were seen in H1299, Calu-1 and
H441 cells, while A549 and H23 cells expressed low levels of Fra-
1. All of the lung cancer cell lines except H441 expressed moderate
to high levels of Fra-2, while only low levels of c-Fos were seen in
all of the lines.
To test whether the c-Jun, Fra-2 and Fra-1 AP-1 subunits play a
role in Blimp1 expression, a knockdown strategy was employed.
The c-Jun subunit can form homodimers with Jun family members
or heterodimers with Fos family members, while Fos family
members only bind as heterodimers with Jun family members
. A549, H441 and H1299 cells were transfected with JUN
siRNA alone or in combination with either FRA-1 or FRA-2
siRNA or a negative control siRNA. A FOS siRNA was not
included as c-Fos expression appeared low in these lines. Effective
knockdown of the corresponding AP-1 subunits in A549, H441
and H1299 cells was confirmed by immunoblot analysis (Fig. 4B).
Depletion of c-Jun alone led to an average 51%, 29% and 23%
decrease in Blimp1 expression in A549, H441 and H1299 cells,
respectively. Simultaneous knockdown of c-Jun and Fra-1 led to
more substantial decreases in Blimp1 expression of 80%, 55% and
40% in A549, H441 and H1299 cells, respectively. Knockdown of
c-Jun and Fra-2 led to decreases in Blimp1 expression by 78%,
48% and 38% in A549, H441 and H1299 cells, respectively.
These results indicate that AP-1 subunits c-Jun, Fra-1 and Fra-2
are all involved in the maintenance of basal Blimp1 expression in
lung cancer cells.
We next tested whether AP-1 complexes containing c-Jun with
either Fra-1, Fra-2 or c-Fos induce Blimp1 expression and selected
H441 cells, which express a low endogenous level of c-Jun. H441
cells were transfected with c-Jun, Fra-1, Fra-2 or c-Fos cDNA
individually or in combination or with EV DNA for 48 h and
subunit expression confirmed by immunoblotting (Fig. 5A, lower
panels). Q-PCR was performed to measure the effects on BLIMP1
mRNA expression. Data from three independent experiments
show that relative to EV DNA, which was set to 1.0, ectopic
expression of c-Jun, c-Jun-Fra-1, c-Jun-Fra-2 or c-Jun-c-Fos
induced BLIMP1 mRNA levels in H441 cells by an average of
2.8-, 2.1-, 1.7 or 2.6-fold, respectively (Fig. 5A, upper panels). Fra-
1, Fra-2 and c-Fos alone had little effect on BLIMP1 mRNA
expression. Immunoblot analyses were quantified to assess the
effects of these AP-1 subunits on Blimp1 protein and the average
values from two independent transfection experiments were
calculated relative to the EV DNA (Fig. 5A, middle panels).
Ectopic expression of c-Jun alone induced endogenous Blimp1
protein expression 3.3-fold and the combinations of c-Jun with
Fra-1, Fra-2 or c-Fos induced Blimp1 by an average of 5.4-, 3.8- or
5.6-fold, respectively. Fra-1, Fra-2 and c-Fos alone were again less
effective (Fig. 5A, darker exposure, middle panels). Thus, AP-1
complexes containing c-Jun with Fra-1, Fra-2 or c-Fos, and
possibly c-Jun homodimers, induce Blimp1 protein and mRNA
expression in lung cancer cells.
Lastly, AP-1 factors were tested for their ability to induce Blimp1
promoter activity in H441 lung cancer cells using a co-transfection
assay. c-Jun alone or in combination with Fra-1, Fra-2 or c-Fos
substantially induced normalized Blimp1 promoter activity by an
average of 6.4-, 3.6-, 7.7- or 5.1-fold, respectively (Fig. 5B).
Expression of Fra-1, Fra-2, or c-Fos cDNA alone had only minor
effects on the activity of the Blimp1 promoter, as expected.
Together, these results show that c-Jun containing AP-1 complexes
(c-Jun-c-Jun, c-Jun-Fra-1, c-Jun-Fra-2 and c-Jun-c-Fos effectively
induce Blimp1 promoter activity, leading to elevated levels of
Ectopic c-Jun binds to the BLIMP1 promoter
Next, binding of AP-1 subunits to the BLIMP1 promoter was
examined using ChIP assays. The two identified AP-1 binding sites
are located at 21647 and 21813 bp relative to the BLIMP1
transcription start  (Fig. 6A). Since c-Jun plays an essential role
in formation of homo- and hetero-dimers of AP-1 complexes, we
first tested for direct binding of c-Jun to the TRE sites. H441 cells
were transiently transfected with a c-Jun cDNA expression vector.
ChIP analysis was performed using an anti-c-Jun or control IgG
antibody, and resulting genomic DNA fragments analyzed by
PCR (Fig. 6B). Amplification of the 21647 and 21813 bp TRE
sites was observed using the two primer sets indicated in Fig. 6A.
As a positive control, a primer set for previously described TREs
on the JUN promoter was used . Ectopically expressed c-Jun
was also present on its own promoter (Fig. 6B), as expected. As a
negative control, an upstream region of the BLIMP1 promoter
(25508 to 25366 bp) without any known TRE consensus
sequence was tested, and no amplification was observed following
c-Jun antibody pull-down (Fig. 6B). These results indicate that the
ectopically expressed AP-1 c-Jun subunit is recruited to the
BLIMP1 promoter in H441 lung cancer cells.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
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Endogenous AP-1 subunits bind to the BLIMP1 promoter
Our attempts to test for the binding of endogenous c-Jun to the
BLIMP1 promoter in A549 lung cancer cells cultured in growth
medium supplemented with 10% FBS were unsuccessful as no
amplification of either the BLIMP1 or positive control JUN TRE
sites was detected in ChIP analyses (data not shown). Since AP-1
activities are controlled by Ras-MAPK signaling and because AP-
1 subunits have been shown to be recruited to target gene
promoters upon serum stimulation [58,59], we tested the temporal
induction of BLIMP1 mRNA levels, and AP-1 expression as a
function of time after serum stimulation. A549 lung cancer cells
were incubated for 48 h in serum free DMEM medium. FBS was
added back and proteins and mRNA isolated after 0, 15, or
30 min or 1, 2, or 4 h. The expression of AP-1 subunits was
monitored. Phospho-c-Jun levels began to increase by the 15 min
time point and peaked at 2 h, while a substantial increase in total
c-Jun levels was noted at 1 h (Fig. 6C, lower panels). A markedly
slower migration of Fra-2 bands was noted by 15 min, presumably
active phosphorylated forms, which lasted until 30 min. An
increase in the slower migrating, presumably phosphorylated,
Fra-1 was also seen at 15 min, and these levels remained high
throughout the time course. An increase in total Fra-1 levels was
initially observed at the 1 h time point. Levels of c-Fos remained
relatively low but increased at 1 h. Thus, serum rapidly induces
AP-1 phosphorylation and a later increase in total AP-1 expression
levels. RNA, which isolated over the same time course, was
subjected to Q-PCR assays for BLIMP1 and GAPDH, as loading
control. The normalized levels of BLIMP1 mRNA increased
within 15 min after serum stimulation, peaked at ,2-fold at
30 min and stayed elevated until 2 h (Fig. 6C, upper panel). By the
4 h time point, BLIMP1 mRNA levels were low, suggesting a rapid
but transient transcriptional activation. The 30 min time point was
selected to assess for induction of AP-1 binding to the BLIMP1
Figure 4. Knockdown of AP-1 subunits decreases Blimp1 expression in lung cancer cells. (A) The immunoblot of nuclear extracts from
lung cancer cells in Fig. 1A was stripped and re-probed to assess expression of the AP-1 subunits c-Jun, Fra-1, Fra-2 and c-Fos. (B) A549, H441 and
H1299 cells were transfected with 20 nM of JUN siRNA alone or 10 nM of JUN siRNA in combination with 10 nM of FRA-1 or FRA-2 siRNA or with
20 nM of a negative control siRNA (Qiagen) for 24 h. WCE (30 mg) were subjected to immunoblotting for Blimp1, c-Jun, Fra-1, Fra-2 and a-tubulin, as a
loading control. The Blimp1 bands were quantified and normalized to a-tubulin expression, and average values from two independent experiments
presented relative to control siRNA, set to 1.0.
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To test whether the rapid induction of AP-1 activity is
responsible for the increase of BLIMP1 mRNA, A549 cells were
serum deprived, and then stimulated with FBS for 30 min. ChIP
analysis was performed using antibodies against c-Jun, Fra-1, or
Fra-2 or the pre-immune IgG. PCR amplification of the 21647
and 21813 bp TRE sites of the BLIMP1 promoter was observed
with c-Jun, Fra-1 or Fra-2 antibodies (Fig. 6D). The positive
control TRE sites on the JUN promoter were also amplified with
the antibody against c-Jun, consistent with the literature . We
were unable to detect precipitation of the JUN DNA with Fra-1 or
Fra-2 antibodies. No amplification of the negative control region
of the BLIMP1 promoter was observed. Taken together, these
experiments show that endogenous c-Jun, Fra-1 and Fra-2 AP-1
subunits are recruited to the BLIMP1 promoter upon serum
stimulation, further implicating binding of AP-1 subunits to the
TRE sites in regulation of Blimp1 expression.
LOX-PP reduces the expression of Blimp1 and its
upstream AP-1 activators
As the amino terminal LOX-PP domain of Pro-LOX has the
ability to inhibit Ras-mediated transformation of NIH 3T3 cells
 and H1299 lung cancer cells , its ability to reduce Blimp1
was next examined. We first tested the effects of induction of
LOX-PP in two stable H1299 tet-on clones expressing ectopic
LOX-PP, which were described previously . A robust decrease
in BLIMP1 RNA (,90%) was observed upon induction of LOX-
PP, as judged by Q-PCR (Fig. 7A). To extend the findings to a
second line, stable A549 cell populations expressing either human
LOX-PP (hLOX-PP) or mouse LOX-PP (mLOX-PP) in a dox-
inducible vector or with EV DNA were prepared. Following
incubation in the presence of dox for 48 h, induction of ectopic
human or mouse LOX-PP expression was found to reduce
BLIMP1 mRNA by an average of ,60% compared to EV DNA
(Fig. 7B). Ectopic LOX-PP expression was confirmed by
immunoblotting (Fig. 7B). The effects of LOX-PP on Blimp1
protein expression were further analyzed by transiently transfect-
ing A549 and H1299 cells with LOX-PP cDNA. Ectopic
expression of LOX-PP was confirmed by immunoblotting
(Fig. 7C). An average decrease of 53 and 39% in Blimp1
expression was observed in A549 and H1299 cells, respectively.
We next assessed the ability of LOX-PP to reduce the
induction of AP-1 subunits and Blimp1 mediated by serum.
H441 and A549 cells were serum starved for 24 h in culture
medium with 0.5% FBS in the presence of 1 or 4 mg/ml purified
functionally active recombinant LOX-PP protein (rLOX-PP),
respectively, prepared as reported previously [34,60]. FBS was
added back to a final concentration of 10% and cultures
incubated for another 16 h (Fig. 7D). Notably, the levels of
active phospho-c-Jun were decreased by an average of 41% and
34% by LOX-PP treatment in A549 and H441 cells, respectively.
Total c-Jun was decreased by an average of 35% and 50%,
respectively in the LOX-PP-treated A549 and H441 cells. Fra-1
expression was decreased by an average of 30–32% and an
average decrease of 33–35% in Fra-2 expression was observed in
these two cell lines. A commensurate decrease in Blimp1 levels
resulted from LOX-PP treatment (,40% in A549 and H441
cells) (Fig. 7D). Thus LOX-PP reduces the increase in Blimp1
expression and its upstream activators c-Jun, Fra-1 and Fra-2
following serum stimulation.
Figure 5. Ectopic AP-1 subunits induce Blimp1 expression. (A)
H441 cells, growing in 6-well plates, were transfected with 1 mg of
vectors expressing the indicated AP-1 subunits or EV DNA (see bottom)
to make a 2 mg total. Upper panel. After 48 h, RNA was isolated and
subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH
mRNA are presented as mean 6 SD of three independent experiments.
Middle and lower panels. WCE were isolated and subjected to
immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1,
Fra-2, c-Fos and b-actin (Lower panels). (L exp., longer exposure; S exp.,
shorter exposure). Blimp1 levels, normalized to b-actin, were deter-
mined as in Fig. 1C and average values from two independent
experiments presented relative to EV DNA, set to 1.0. (B) H441 cells
were transiently transfected, in triplicate, with 0.3 mg of Blimp1-Luc,
0.3 mg of MSV-b-gal, and vectors expressing the indicated AP-1 subunits
(0.15 mg each) and EV DNA to a total of 1.0 mg DNA. Normalized values
of Blimp1 promoter activity are presented as the mean 6 SEM from two
experiments (EV DNA set to 1.0).
Ras/c-Raf to AP-1 Pathway Activates Blimp1
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LOX-PP-mediated reduction in lung cancer cell migration
occurs via repression of Blimp1
To test the effects of LOX-PP on lung cancer cell migration,
A549 and H441 cells were transiently transfected with a LOX-PP
expressing cDNA or EV DNA. After 24 h, cells were subjected to
a migration assay. LOX-PP expression led to an average decrease
of 39% and 26% in migration of A549 and H441 cells,
respectively, compared to EV (Fig. 8A and 8B, upper panels).
Expression of LOX-PP in the media was confirmed by
immunoblot analysis (Fig. 8A and 8B, lower panels). Similar
results were also observed with addition of rLOX-PP, which
resulted in an ,40% decrease in migration (data not shown).
Next, in order to assess the role of Blimp1 in LOX-PP-
mediated decrease in lung cancer cell migration, we asked
whether ectopic Blimp1 expression can override the observed
inhibition. H441 cells were transiently transfected with EV DNA
or LOX-PP in the absence or presence of Blimp1 cDNA and
subjected to migration assays. Ectopic LOX-PP decreased
endogenous Blimp1 expression and led to a 26% reduction in
cell migration while ectopic Blimp1 increased cell migration by
43% (Fig. 8C), consistent with the earlier findings. Importantly,
LOX-PP-transfected cells expressing ectopic Blimp1 displayed an
ability to migrate at a level similar to those expressing ectopic
Blimp1 only. Immunoblot analysis confirmed ectopic expression
of LOX-PP and Blimp1 expression (Fig. 8D). Together these
results argue for a role of repression of Blimp1 expression in the
inhibition of migration by LOX-PP.
Figure 6. AP-1 subunits bind to the BLIMP1 promoter. (A) Schematic of the localization of the two TRE sites on the human BLIMP1 promoter.
Two primer sets encompassing these sites are shown: 1F/1R amplifies the 21647 bp TRE and 2F/2R amplifies the 21813 bp TRE. (B) H441 cells at
90% confluence in P100 plates were transfected with 4 mg of c-Jun expression vector, and after 48 h subjected to a ChIP assay using a control IgG or
c-Jun antibody, as described in the Materials and Methods. Input, 1% of the WCE. Positive (Pos.) control: a genomic region of the JUN promoter
containing two TREs (21 and 2120 bp). Negative (Neg.) control: region upstream of BLIMP1 transcription start site (,25.4 kB) that does not contain
any known TRE sites. (C) A549 lung cancer cells were incubated in serum free DMEM for 48 h. FBS was added back to 10%. Samples were harvested at
0, 15, 30 minutes or 1, 2, 4 h and RNA and WCE prepared. Upper panel: RNA was subjected to Q-PCR, in triplicate, and values for BLIMP1 normalized
to GAPDH RNA levels presented relative to the 0 time point which was set to 1.0. Data for the mean 6 SD from three independent experiments are
presented. Lower panels: WCE (25 mg) were subjected to immunoblot analysis for phospho-c-Jun (p-c-Jun), c-Jun, Fra-1, Fra-2, c-Fos AP-1 subunits,
and a-tubulin, which confirmed essentially equal loading control. Data shown is a representative of two independent experiments with similar results.
(D) A549 cells were incubated in serum free DMEM for 48 h, and stimulated with addition of FBS (final 10%) for 30 min. Whole cell lysates were
subjected to ChIP analysis using antibodies against c-Jun, Fra-1, Fra-2 or normal rabbit IgG, as described in part B. Data shown is a representative of
two independent experiments with similar results.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
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Physical interaction of LOX-PP with c-Raf inhibits Blimp1-
mediated cell migration
We recently noted that LOX-PP can physically interact with c-
Raf and Hsp70 in breast cancer cells . Given the role of c-Raf
in activation of Blimp1, their physical association in lung cancer
cells was next tested. Association of c-Raf with LOX-PP in the
H1299 lung cancer line was monitored using GST-pull down
assays. Cells were transfected with a vector expressing LOX-PP-
GST or GST protein. LOX-PP-GST brought down c-Raf and
Hsp70, but not B-Raf, Hsp90, Erk1/2 and MEK1/2 (Fig. 9A).
Triton X-100 soluble lysates of A549 cells were immunoprecip-
itated with an antibody against either LOX-PP or rabbit control
IgG. The antibody against LOX-PP brought down the LOX-PP
peptide as well as c-Raf, confirming the ability of these
Figure 7. Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7
(PP7) clones, isolated as described previously , were treated in triplicate with 2 mg/ml dox for 48 h. RNA from two independent experiments was
subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean 6 SEM (EV DNA set to 1.0). (B) A549-
EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 mg/ml dox for 48 h in DMEM supplemented with 0.5% FBS.
FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values
for BLIMP1 mRNA relative to GAPDH levels are presented as the mean 6 SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to
immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected
with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 ml) were subjected to immunoblotting for V5.
Samples of WCE (25 mg) were probed for Blimp1 and b-actin, and average normalized Blimp1 values from two independent experiments presented
relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 mg/ml,
respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated
overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and a-tubulin, as a loading
control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33287
endogenous proteins to interact (Fig. 9B). The region of LOX-PP
encompassing aa 26 to 100 is necessary for its interaction with c-
Raf in breast cancer cells . Lysates were prepared from H1299
cells ectopically expressing either GST, GST-LOX-PP WT or
GST-LOX-PP DM3 (with a deletion of aa 26–100) and subjected
to purification with Glutathione-Sepharose 4B beads. Binding of c-
Raf to full-length LOX-PP was readily detected, whereas no
binding was seen with the GST-LOX-PP DM3 protein, or with
the GST control protein (Fig. 9C). These results suggest that aa 26
to 100 of LOX-PP is necessary for its interaction with c-Raf in
H1299 cells. To assess the role of LOX-PP and c-Raf interaction
in migration, assays were performed 24 h after ectopic expression
of GST, full-length GST-LOX-PP WT, or GST-LOX-PP DM3 in
H1299 cells. An approximately 40% reduction was seen with
expression of full-length LOX-PP. In contrast, no reduction in
H1299 cell migration was seen with the LOX-PP DM3 mutant
(Fig. 9D). Lastly, a constitutively active mutant of MEK, which is
downstream of c-Raf, was able to override the inhibition of
migration by LOX-PP (Fig. 9E), confirming the importance of this
pathway. Thus, the domain comprising aa 26 to 100, which
mediates the interaction of LOX-PP with c-Raf, is required for
inhibition of migratory activity.
Here we demonstrate that Blimp1, the zinc finger master
regulator of B and T cells, is aberrantly activated in lung cancer
cells by the oncogenic Ras/c-Raf to AP-1 pathway, and functions
to promote their migratory phenotype. Furthermore, high
BLIMP1 RNA typifies several other aggressive cancers frequently
driven by Ras signaling, including pancreatic and head and neck
carcinomas as well as glioblastomas. The anti-cancer peptide
LOX-PP, which was found to interact with c-Raf in lung cancer
cells, repressed the induction of AP-1. Blimp1, which was detected
in all five lung cancer cell lines examined, promoted lung cancer
cell migration as judged by both knockdown and ectopic
expression approaches. The ability of a dominant-negative Ras
(Ras S186), and of knockdown of K-Ras and c-Raf and multiple
AP-1 subunits to decrease Blimp1 levels in lung cancer cells
confirmed the role of this signaling axis in aberrant expression of
this zinc finger protein. Notably, c-Raf has recently been found
essential for development of K-Ras-driven NSCLCs . The
ability of LOX-PP to inhibit this pathway and the observation that
lung cancers are typified by greatly reduced levels of LOX gene
expression [24,25], suggests the potential use of LOX-PP in
therapy of these cancers.
Mechanistically, Blimp1 expression is shown here to be
positively regulated by AP-1 subunits. To our knowledge, this is
the first study showing the induction of Blimp1 expression by AP-1
factors in epithelial cancer cells. Of note, deregulated AP-1 activity
alone is sufficient for neoplastic transformation and critically
necessary for the function of upstream dominant oncogenes,
including members of growth factor receptor family which signal
via the Ras-MAPK system . AP-1 subunits including c-Jun, c-
Fos and Fra-1 have been implicated in promoting cell motility in
lung cancer [20,64,65]; although, the downstream targets were not
fully elucidated. The c-Fos AP-1 subunit was shown to induce
accelerated expression of Blimp1 upon CD40L/IL-4 treatment of
B cells . Our data implicate c-Jun containing complexes (c-
Jun-c-Jun, c-Jun-Fra-1, c-Jun-Fra-2 and c-Jun-c-Fos) in Blimp1
expression in lung cancer cells. The observation that c-Jun, Fra-1
and Fra-2 AP-1 subunits are physically present on the TRE sites of
the BLIMP1 promoter indicates their direct control of transcrip-
tion following growth factor stimulation. Activation of ERK has
Figure 8. LOX-PP represses the migratory phenotype of lung
cancer cells via inhibiting Blimp1. (A) A549 and (B) H441 cells were
transfected with 2 mg of EV or human LOX-PP cDNA. Upper panels:
After 24 h, 16105transfected cells were subjected to migration assay.
Lower panels: After 48 h, culture media was isolated and samples (50 ml
of 2 ml total) subjected to immunoblotting using an anti-V5 antibody
for LOX-PP. (C and D) H441 were transiently transfected with 1 mg of
LOX-PP or Blimp1 DNA alone or in combination, or EV DNA (2 mg total
DNA). (C) After 24 h. cells were subjected to migration assays, in
triplicate, for 16 h. The average migration from two independent
experiments 6 SEM is presented relative to the EV (set at 1.0). (D) WCE
and media were isolated. WCE samples (25 mg) were subjected to
immunoblotting for Blimp1 and a-tubulin. Media samples (50 ml) were
subjected to immunoblotting for LOX-PP-V5. Immunoblots from one of
two independent experiments with similar results are presented.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
PLoS ONE | www.plosone.org12March 2012 | Volume 7 | Issue 3 | e33287
been found to lead to c-Jun phosphorylation at Ser 63 and 73
[67,68] which subsequently upregulates c-Jun expression via a
positive feed-back loop. In addition, Fra-1 and Fra-2 are directly
activated by ERK which may enhance their DNA binding in
conjunction with c-Jun . These findings suggest that the Ras
downstream effectors c-Jun, Fra-1 and Fra-2 are all involved in the
expression of Blimp1 in lung cancer cells.
Microarray data from Oncomine confirmed upregulated
BLIMP1 mRNA expression is present in lung, pancreatic, head
and neck cancers, and glioblastomas. In addition to oncogenic Ras
mutations, 15–30% of samples of NSCLC, which make up 85% of
total lung cancers, were also found positive for overexpression of
EGFR , which signals via Ras, and has been implicated in
their increased ability to invade and metastasize . It is known
that Ras mutations, especially activating K-Ras mutations, occur
in more than 95% of pancreatic cancers . High frequencies of
EGFR overexpression have been reported in head and neck
squamous cell carcinomas  and glioblastomas . All these
evidences suggest a role for Blimp1 as a mediator of the aberrantly
activated Ras/MAPK signaling pathway under pathological
conditions. Although Blimp1 may play an important etiologic
role in development of these cancers in vivo, unfortunately, as was
found with NF-kB, it cannot likely serve as a direct therapeutic
target for most cancers given its essential role in directing the
immune response. However, we hypothesized that as Blimp1 can
only repress genes that are being actively expressed, that a distinct
subset of targets will exist in transformed epithelial vs immune
cells. Our microarray data in breast cancer cells has confirmed this
hypothesis (Mathilde Romagnoli and G.E.S., unpublished obser-
vations). Together, these findings suggest that the observed
aberrant expression of Blimp1 in lung and other epithelial cancers
may have important clinical ramifications, leading to development
of new therapeutic modalities.
We gratefully acknowledge Tom Maniatis, Kathryn Calame, Tsuyoshi
Akagi, Bruce Mayer and Geoffrey Cooper for providing cloned DNAs, and
Zhi-Xiong Jim Xiao, Hasmeena Kathuria and Maria Ramirez for cell
lines. We also thank Karine Belguise for advice on the functional analysis of
Conceived and designed the experiments: ZY SS KHK PCT GES.
Performed the experiments: ZY SS. Analyzed the data: ZY SS KHK PCT
GES. Wrote the paper: ZY SS GES. Gave final approval: ZY SS KHK
Figure 9. LOX-PP inhibits the migratory phenotype of lung cancer cells via interaction with c-Raf. (A) H1299 cells were transfected with
expression plasmids for GST or LOX-PP-GST (PP-GST) and tagged proteins purified on Glutathione-Sepharose 4B beads. Bound proteins were
analyzed by immunoblotting for the indicated proteins. For estimation of the amounts of expressed proteins, 4% of each of the lysates was
immunoblotted (Input). (B) Triton X-100 extracts of A549 were immunoprecipitated with the indicated antibodies. The precipitated proteins were
analyzed by Western blotting for c-Raf and LOX-PP. As the band of precipitated LOX-PP migrated close to that of rabbit IgG light chain, Protein A-
conjugated HRP was used as a secondary ‘antibody’ to detect immunoprecipitated LOX-PP. (C) GST (EV), GST-LOX-PP WT (G-PP-WT), or GST-LOX-PP
DM3 (G-PP-DM3) were expressed in H1299 cells for 24 h and tagged proteins purified as in part A and subjected to Western blotting with the
indicated antibodies. Input=4% of the lysate. (D) Either GST (EV), GST-LOX-PP WT or GST-LOX-PP DM3 (D26–100) were expressed in H1299. After
24 h, cells were subjected to a migration assay in triplicate for 16 h. Cells that migrated to the lower side of the filter were stained and quantified by
spectrometric determination at A570 nm. The average values from three independent experiments 6 SD presented relative to the EV control, set to 1.0.
P values were calculated from three independent experiments using Student’s t test. *, P,0.01. (E) GST (EV) or GST-LOX-PP (PP) was co-transfected in
H1299 cells in presence of a vector expressing a constitutively active MEK mutant (CA-MEK) or EV DNA (2) for 24 h. Cultures were subjected to a
migration assay for 16 h, in triplicate, as in part D. P values of three independent experiments were calculated using Student’s t test. *, P,0.01.
Ras/c-Raf to AP-1 Pathway Activates Blimp1
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