XB130 Mediates Cancer Cell Proliferation and Survival
through Multiple Signaling Events Downstream of Akt
Atsushi Shiozaki1, Grace Shen-Tu1, Xiaohui Bai1, Daisuke Iitaka1, Valentina De Falco3, Massimo Santoro3,
Shaf Keshavjee1,2, Mingyao Liu1,2*
1Latner Thoracic Surgery Research Laboratories, University Health Network Toronto General Research Institute, Toronto, Ontario, Canada, 2Department of Surgery,
Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada, 3Dipartimento di Biologia e Patologia Cellulare e Molecolare, Istituto di Endocrinologia ed
Oncologia Sperimentale del CNR ‘G. Salvatore’, Naples, Italy
XB130, a novel adaptor protein, mediates RET/PTC chromosome rearrangement-related thyroid cancer cell proliferation and
survival through phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway. Recently, XB130 was found in different cancer cells in
the absence of RET/PTC. To determine whether RET/PTC is required of XB130-related cancer cell proliferation and survival,
WRO thyroid cancer cells (with RET/PTC mutation) and A549 lung cancer cells (without RET/PTC) were treated with XB130
siRNA, and multiple Akt down-stream signals were examined. Knocking-down of XB130 inhibited G1-S phase progression,
and induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Knocking-
down of XB130 reduced phosphorylation of p21Cip1/WAF1, p27Kip1, FOXO3a and GSK3b, increased p21Cip1/WAF1protein
levels and cleavages of caspase-8 and-9. However, the phosphorylation of FOXO1 and the protein levels of p53 were not
affected by XB130 siRNA. We also found XB130 can be phosphorylated by multiple protein tyrosine kinases. These results
indicate that XB130 is a substrate of multiple protein tyrosine kinases, and it can regulate cell proliferation and survival
through modulating selected down-stream signals of PI3K/Akt pathway. XB130 could be involved in growth and survival of
different cancer cells.
Citation: Shiozaki A, Shen-Tu G, Bai X, Iitaka D, De Falco V, et al. (2012) XB130 Mediates Cancer Cell Proliferation and Survival through Multiple Signaling Events
Downstream of Akt. PLoS ONE 7(8): e43646. doi:10.1371/journal.pone.0043646
Editor: Laszlo Buday, Hungarian Academy of Sciences, Hungary
Received March 23, 2012; Accepted July 24, 2012; Published August 2 , 2012
Copyright: ? 2012 Shiozaki 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: This work was supported by operating grants (MOP-13270 and MOP-42546) from Canadian Institutes of Health Research, Research Fellowship Awards
from Uehara Memorial Foundation and International Society of Heart and Lung Transplantation for Dr. Atsushi Shiozaki and by Associazione Italiana Ricerca sul
Cancro (AIRC). 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
XB130 is a newly discovered adaptor protein for intracellular
signal transduction; it is involved in gene regulation, cell pro-
liferation, cell survival, cell migration, and tumorigenesis .
Human xb130 gene was discovered during the cloning process of
human afap gene [2,3,4]. It encodes 818 amino acids with an
apparent molecular size of approximately 130 kDa. The overall
structure of XB130 shares similarity with AFAP thus is also known
as AFAP1 like protein 2 (AFAP1L2). As an adaptor protein, the N-
terminal region of XB130 includes several tyrosine phosphoryla-
tion sites and proline-rich sequence, which can potentially interact
with SH2 and SH3 domain-containing proteins, respectively. The
middle portion harbors two pleckstrin-homology (PH) domains
that may target proteins to cellular membranes through interac-
tions with specific phospholipids. The C-terminal region contains
a coiled-coil domain, which might be involved in protein
oligomerization and DNA binding . XB130 can interact and
activate c-Src tyrosine kinase, leading to elevated tyrosine
phosphorylation of multiple proteins including XB130, and
transactivation of AP-1 and SRE . During cell migration,
XB130 regulates actin cytoskeleton rearrangement as demonstrat-
ed by its translocation to cell periphery in lamellipodia. Silencing
endogenous XB130 can cause a decrease in the rate of wound
closure, inhibit matrigel invasion, and reduce lamellipodial
persistence and cell spreading, which suggest that it plays an
important role in cell motility and invasion .
XB130 is involved in thyroid cancer cell proliferation and
survival . Thyroid cancer is a type of endocrine malignancy,
which involves multiple genetic and epigenetic alterations leading
to MAPK and PI3K/AKT signaling pathway activation [6,7,8]. A
common mutation found in thyroid cancer is RET/PTC
chromosomal rearrangements. The product RET/PTC is a pro-
tein produced from the chromosomal rearrangement with the
combination of the 39 portion of the RET gene and the 59 section
of a partner gene. This results in a constitutively activated tyrosine
kinase, RET/PTC . RET/PTC exhibits transforming ability
via effecting differentiation, mitogenic and metastatic potential in
thyroid cancer [10,11]. RET/PTC causes a robust tyrosine
phosphorylation of XB130, which promotes its association with
the p85a subunit of phosphatidylinositol 3-kinase (PI3K). This in
turn activates Akt. Down-regulation of XB130 in TPC1 papillary
thyroid cancer cells, harboring the RET/PTC kinase, strongly
reduced Akt activity, cell-cycle progression and cell survival .
Furthermore, in WRO cells, another thyroid cancer cell line with
RET/PTC mutation , cells stably transfected with XB130
shRNA reduced tumor growth in nude mice. Microarray study
identified that multiple genes regulated by XB130 are related to
PLOS ONE | www.plosone.org1August 2012 | Volume 7 | Issue 8 | e43646
cell proliferation or survival, including many transcription
Since XB130 is highly expressed in thyroid, it has been
speculated to be a thyroid-specific tyrosine kinase substrate.
Recently, we have found expression of XB130 in esophageal
cancer , and in other cancer cell lines. In the present study we
sought to determine whether XB130 plays a role in cancer cells
independent from the presence of RET/PTC. Furthermore,
although the PI3K/AKT pathway has been identified as
important for XB130-mediated cell proliferation and survival,
the downstream signals of Akt are as yet undetermined. Thus, we
studied these events with WRO cells, a human thyroid cancer cell
line with RET/PTC rearrangement, and A549, a human lung
adenocarcinoma cell line without RET/PTC.
Materials and Methods
Cell Lines, Antibodies and Other Reagents
Human follicular thyroid carcinoma WRO cells (established by
Dr GJF Juillard, University of California-Los Angeles School of
Medicine, Los Angeles, CA, USA) were maintained in RPMI
1640, supplemented with 10% FBS, 1 mM pyruvate and non-
essential amino acids (GIBCO-BRL, Gaithersburg, MD, USA)
. Human lung adenocarcinoma A549 cells, obtained from
ATCC (CCL-185; Manassas, VA) , were grown in DMEM
medium, supplemented with 10% FBS, 1% penicillin-streptomy-
cin, and 1% glutamine. Cells were cultured in a standard
humidified incubator at 37uC with 5% CO2.
Expression vectors for RET/PTC3, activated versions of ABL
and SRC , or EGFR and ERBB2 , which are activated
upon overexpression, are described elsewhere. Monoclonal XB130
antibody was generated as described previously . Antibodies for
phospho-Akt (Ser473), Akt, phospho-GSK-3b (Ser9), p21Cip1/
WAF1, p27Kip1, p53, phospho-FoxO3a (Thr32), FoxO3a,
phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phospho-
p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-p44/42
MAPK (Thr202/Try204), phosho-Src (Try416) were from Cell
Signaling Technology (Beverly, MA, USA); monoclonal anti-
phosphotyrosine antibodies were from Upstate Biotechnology Inc.
(Lake Placid, NY, USA) and anti-c-myc tag (sc-40) were from
Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for
GAPDH, ERK1, phospho-p21 (Thr145), caspase-8 and caspase-9
were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-PCNA antibody was from Abcam (Cambridge, MA, USA).
Anti-Ki-67, clone Ki-S5, was from Chemicon (Temecula, CA,
USA). Anti-Src (GD11) and anti-Fas mAb (clone CH11) were
from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Anti-
Phospho-p27Kip1 (Thr157) antibody was from R&D Systems Inc.
(Minneapolis, MN, USA). Anti-p85a of PI3K antibody was from
BD PharMingen (San Diego, CA, USA). Staurosporine was from
Sigma-Aldrich (St. Louis, MO, USA).
Immunoblotting experiments were performed according to
standard procedures described previously [2,3]. Briefly, Cells were
lysed with modified radioimmune precipitation assay buffer
(50 mM Tris-HCl; pH 7.5, 150 mM NaCl, 2 mM EGTA,
2 mM EDTA and 1% Triton X-100) containing 10 mg/ml each
aprotinin, leupeptin, pepstatin, 1 mM phenylmethylsulfonyl fluo-
ride, 1 mM Na3VO4, and 10 mM NaF. Protein concentration was
measured with a modified Bradford assay (Bio-Rad, Munich,
Germany). Cell lysates containing equal amount of total proteins
were separated by SDS-PAGE and then transferred onto
nitrocellulose membranes (Schleicher & Schuell, Whatman,
Middlesex, UK). Membranes were then probed with the indicated
antibodies. Proteins were revealed by an enhanced chemilumines-
cence detection kit (ECL) (Amersham Pharmacia Biotech, Little
Chalfort, UK). Band densities were quantified using the ImageJ
software (http://rsb.info.nih.gov/ij/) after being scanned from the
film. The protocol for immunoprecipitation has been described
previously in detail [3,19].
Two siRNAs were designed according to XB130 sequence as
described previously [4,12]. Cells were transfected with 50 nM
pooled XB130 siRNAs using the oligofectamine reagent (Invitro-
gen, San Diego, CA, USA) according to standard procedures
described previously [4,20]. The medium containing siRNA was
replaced with fresh medium after 24 h. The siSTABLE V2 non-
targeting siRNA#1 from Dharmacon (Lafayette, CO, USA) was
used as a negative control. For protein studies, siRNA transfected
cells were harvested at 48 h after transfection.
Real-time Quantitative RT-PCR
Sequence-specific primers for human RET/PTC were designed
by Balogh et al.: 59-GTCGGGGGGCATTGTCATCT-39 and
59-AAGTTCTTCCGAGGGAATTC-39 . Total RNA was
extracted using RNeasy kit (Qiagen, Valencia, CA), and cDNA
was synthesized from total RNA using MuLV Reverse Transcrip-
tase (Applied Biosystems, Carlsbad, CA). Quantitative RT-PCR
was performed using SYBR Green I Master PCR kit on
LightCycler480 (Roche, Indianapolis, IN). Each assay included
a standard curve of five serial dilutions and a no-template negative
control. All assays were performed in triplicate. Gene expression
levels were normalized to the level of SDHA, a housekeeping gene
Cell Cycle Analysis
Cell cycle distribution was analyzed by flow cytometry .
Briefly, cells were harvested in phospho-buffered saline (PBS), and
fixed in 70% cold ethanol overnight. Fixed cells were washed twice
in PBS and permeabilized with 0.1% Triton X-100 and 2 mg/ml
RNase A in PBS for 30 min. They were then washed once in PBS
and stained with 50 mg/ml of propidium iodide (PI) (Sigma-
Aldrich). Stained cells were analyzed with the Cytomix FC500
flow cytometry system (Beckman-coulter, Fullerton, CA, USA).
Analysis of Apoptotic Cells
After treated with staurosporine or FasAb for 24 h, cells were
harvested and stained with flourescein isothiocyanate (FITC)-
conjugated Annexin V and PI using the Annexin V kit (Beckman
Coulter, Brea, CA, USA) following manufacturer’s protocols and
analyzed by the Cytomix FC500 flow cytometry system.
Statistical analysis was carried out using Student’s t-test.
Differences were considered significant when the P value was less
than 0.05. Statistical analyses were performed using the statistical
software JMP version 5 (SAS Institute Inc., Cary, NC, USA).
XB130 Controls Cell Cycle Progression of Cancer Cells
To explore the role of XB130 in cancer cell cycle progression
we conducted knockdown experiments using XB130 siRNA in
human thyroid follicular carcinoma WRO cells (with RET-PTC
rearrangement) and in human lung adenocarcinoma A549 cells
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org2August 2012 | Volume 7 | Issue 8 | e43646
(a cell line commonly used in lung cancer studies ). Two
siRNAs targeting different sites of XB130 were designed. We
have previously shown that both of them effectively reduced
XB130 protein levels and Akt phosphorylation in TPC-1 cells
 and other cell types (data not shown). Combined application
of these two siRNAs reduces the concentration of each siRNA to
half, thus may reduce potential off-target effects. Therefore, we
have used this strategy previously [5,12] and also in the present
studies. Flow cytometry showed that down-regulation of XB130
partially blocked cell cycle progression from G1to S phase in
both WRO and A549 cells as determined by flow cytometry
(Fig. 1A). There are significantly more cells in G1 phases than in
either the S or G2/M phase (Fig. 1B). While, western blots
illustrate a significant reduction in cell proliferation markers, Ki-
67 and PCNA, in the XB130 siRNA treated cells (Fig. 1C). The
presence of RET-PTC rearrangement was confirmed in TPC-1
cells (as a positive control) and in WRO cells, whereas it is
absence in A549 cells as shown by RT-PCR (Fig. 1D). These
results indicate that XB130 plays important roles in the
regulation of cancer cell proliferation either in the presence or
the absence of RET/PTC.
XB130 Controls Survival of Cancer Cells
To determine the role of XB130 in cancer cell survival, we
treated WRO and A549 cells with XB130 siRNA and used flow
cytometry to quantify and to discriminate between the extrinsic
(death receptor mediated) apoptotic pathway, and the intrinsic
(mitochondrial mediated) apoptotic pathway . Down-regula-
tion of XB130 induced early apoptosis (Annexin V positive/PI
negative) in WRO cells at 48 h after transfection of siRNA
(Fig. 2A). Further, XB130 siRNA enhanced extrinsic (FasAb, clone
CH11, 100 ng/ml) and intrinsic (staurosporine, 200 nM) apopto-
tic stimulus-induced early and late apoptosis (Annexin V/PI
double positive) (Fig. 2A). On the other hand, in A549 cells
cultured in the medium containing 10% FBS, down-regulation of
XB130 did not enhance spontaneous nor the staurosporin-or
FasAb-induced cell death, even at 500 ng/ml of FasAb (Fig. S1A).
Further, combined use of FasAb (500 ng/ml) and IFN-c (100 ng/
ml), a condition that has been previously reported to induced cell
death in several other types of cells [24,25], did not induce
apoptosis in A549 cells (Fig. S1B). Western blotting revealed that
the expression of endogenous Fas in A549 cells is even higher than
that in WRO cells (Fig. S1C). However, under serum free
condition, XB130 siRNA treatment enhanced staurosporine-
induced early apoptosis in A549 cells (Fig. 2B). These findings
indicate that A549 cells are resistant to both extrinsic and
intrinsically mediated apoptotic signals. Nevertheless, XB130 is
involved in cell survival in both cell lines under divergent
Even though A549 cells do not have RET/PTC, other
endogenous PTKs could phosphorylate XB130. Accordingly, we
have shown dramatic protein tyrosine phosphorylation in A549
cells, which can be effectively reduced by SRC inhibitor, PP2 .
To test whether whether other protein kinases could phosphor-
ylate XB130 in tyrosine, HEK293 cells, a cell line commonly used
to test protein-protein interaction, were transfected with myc-
tagged XB130 together with RET/PTC3 as a control, or other
membrane-bound (EGFR, ERBB2) or cytosolic (SRC, ABL)
tyrosine kinases. Cell lysates were immunoprecipitated with anti-
myc antibody and blotted with anti-phosphotyrosine antibody.
Though at different levels, all the protein tyrosine kinases co-
expressed with XB130 increased its tyrosine phosphorylation
XB130 Binds to p85a Subunit of PI3K and Controls Akt
Activity in Cancer Cells
The two well known signaling cascade that are implicated in the
regulation of cell progression and survival via protein phosphor-
ylation are the PI3K/Akt and p38 MAPK pathways . We tested
key signaling components in these two pathways and found that
the phosphorylation Src, JNK and ERK was not affected in
XB130 siRNA treated cells (Fig. S2). Moreover, the phosphory-
lation of p38 was even significantly increased after XB130 siRAN
treatment in WRO cells (Fig. S2), which further exclude the
possibility of p38 as a down-stream signal that mediates XB130-
related cell proliferation and survival.
We have previously shown that XB130 regulate thyroid cancer
cell cycle progression and survival through its interaction with
PI3K, leading to the activation of Akt. XB130 has an YxxM motif
in the N-terminus starting at the tyrosine 54 that can bind to either
the N-or the C-terminal SH2 domain of p85a-subunit of PI3K
(Fig. 3A), as demonstrated by GST-fusion protein pull-down
assays, co-immunoprecipitation, phosphor-peptide competition,
and use of XB130 mutants . Recently, the binding between
XB130 and p85 was also reported by Yamanaka et al. in rat
thyroid FRTL-5 cells with MOLDI-TOF MS assay . In both
WRO and A549 cells, binding between XB130 and p85a subunit
of PI3K was shown by co-immunoprecipitation (Fig. 3A). Akt is
a downstream target of PI3K, and its phosphorylation at Ser 473 is
a sensitive indicator of Akt activity . XB130 siRNA effectively
reduced XB130 protein levels; a phenomenon associated with
decreased phosphorylation of Akt in both WRO and A549 cells
XB130 Controls Cancer Cell Cycle Progression and
Survival via Multiple Akt Down Stream Molecules
p27Kip1 and p21Cip1/WAF1 are involved in the regulation of
cell cycle progression through the inhibition of cyclin-dependent
kinases CDK1 and CDK2. The cyclin-dependent kinase inhibitor
p27Kip1 nuclear translocation can cause G1 arrest; Akt is able to
phosphorylate p27Kip1, and thus block its translocation and
function [29,30,31]. The cyclin-dependent kinase inhibitor
p21Cip1/WAF1 can negatively modulate cell cycle progression
by inhibiting the activation of cyclin/cdk2 complex .
Activation of Akt can also phosphorylate p21Cip1/WAF1 and
keep it in the cytoplasm, which may be critical for cell survival
. After XB130 siRNA treatment, the phosphorylation of
p27Kip1 and p21Cip1/WAF1 were significantly reduced in both
WRO and A549 cells (Fig. 4A and B).
Down-regulation of XB130 also significantly increased the total
protein level of p21Cip1/WAF1 (Fig. 4A and B). FOXO3a can
bind to and activate the promoter of p21Cip1/WAF1, which has
forkhead binding elements adjacent to a SMAD-binding element.
PI3K/Akt mediated FOXO3a phosphorylation may lead to its
export from the nucleus and subsequently prevent p21Cip1/
WAF1 gene expression . The XB130 knockdown reduced the
phosphorylation of FOXO3a (Thr32), without affecting the total
FOXO3a levels (Fig. 4C and D). This decreased phosphorylation
of FOXO3a may help to increase the p21Cip1/WAF1 expression.
Among FOXO family members, FOXO1 is known to stimulate
transcription of p27Kip1 . The XB130 knockdown was unable
to change the phosphorylation of FOXO1 (Thr24) and FOXO1
(Ser256) (data not shown). Coincidently, XB130 siRNA treatment
had no effect on p27Kip1 protein levels in both cell lines (Fig. 4A
Another mechanism by which Akt can promote cell pro-
liferation is to phosphorylate and inactivate GSK3b, thus
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org3August 2012 | Volume 7 | Issue 8 | e43646
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org4 August 2012 | Volume 7 | Issue 8 | e43646
Figure 1. XB130 controls cell cycle progression of cancer cells. (A and B) Down-regulation of XB130 inhibited G1-S phase progression in WRO
and A549 cells. Cells transfected with control or XB130 siRNA were stained with PI and analyzed by flow cytometry. Mean 6 SEM. n=6. *p,0.05
(compared with control siRNA). (C) Reduced Ki67 and PCNA levels in XB130 siRNA treated WRO and A549 cells, as determined by western blotting.
n=3. *p,0.05 (compared with control siRNA treated group). (D) Presence of RET/PTC rearrangement was confirmed in both TPC-1 and WRO cells
using RT-PCR. A549 cells do not contain RET/PTC rearrangement.
Figure 2. XB130 controls cell survival of cancer cells. (A) Down-regulation of XB130 enhanced spontaneous and induced cell death of WRO
cells cultured with 10% FBS. Cells transfected with control or XB130 siRNA were treated with staurosporine (STS, 200 nM), or Fas antibody (FasAb,
clone CH11, 100 ng/ml) for 24 h. Apoptosis was determined by flow cytometry using PI/Annexin V double staining. (B) Down-regulation of XB130
enhanced staurosporine-induced apoptosis in serum free condition in A549 cells. A549 cells transfected with control or XB130 siRNA were incubated
in serum free medium with or without 200 nM STS for 24 h. n=6. Mean 6 SEM. *p,0.05 (compared with control siRNA). (C) XB130 can be
phosphorylated by protein tyrosine kinases other than RET/PTC. Immunoprecipitation with anti-myc antibody in HEK293 cells transfected with myc-
tagged XB130, along with RET/PTC3, EGFR, Src, Abl, or ERBB2, showed an increase in XB130 tyrosine phosphorylation using anti-phosphotyrosine
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org5 August 2012 | Volume 7 | Issue 8 | e43646
protecting cyclin D1, enhancing CDK4/CDK6 activity, and
facilitate cell entry into S phase of the cell cycle .
Phosphorylation of GSK3b was decreased after XB130 siRNA
treatment (Fig. 4C and D). The tumor suppressor protein p53 is
a transcription factor that can induce either growth arrest or
apoptosis. Its levels and activities are mainly controlled by MDM2,
which can bind p53 directly and promote its ubiquitination and
degradation. Akt can phosphorylate MDM2 and thus increase p53
degradation . Down-regulation of XB130 with siRNA,
however, did not affect p53 levels.
Akt can inhibit apoptosis through multiple mechanisms. For
example, Akt is able to phosphorylate procaspase-9, preventing its
cleavage into the pro-apoptotic caspase-9, which mediates intrinsic
signal initiated apoptosis . Inhibition of Akt enhanced TRAIL-
induced activation of caspase-8, which mediates extrinsic signal
initiated apoptosis . Down-regulation of XB130 increased the
cleavage of caspase-8 and caspease-9 in WRO cells (Fig. 5A and
B). In A549 cells, down-regulation of XB130 decreased procas-
pase-8 level and increased cleaved caspase-9 (Fig. 5A and B).
Activation (cleavage) of caspase-8 and caspase-9 are essential steps
for extrinsic and intrinsic pathways of cell death, respectively .
Figure 3. XB130 binds to p85a subunit of PI3K and controls Akt activity in cancer cells. (A) Schematic representation of protein structures
of XB130 and p85a subunit of PI3K. Interactions of XB130 with p85a were detected by co-immunoprecipitation in WRO and A549 cells. (B) XB130
siRNA effectively reduced XB130 protein levels and Akt phosphorylation in WRO and A549 cells. siRNA transfected cells were harvested at 48 h after
transfection. n=5. *p,0.05 (compared with control siRNA).
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org6 August 2012 | Volume 7 | Issue 8 | e43646
Figure 4. XB130 controls cell cycle progression and survival via PI3K/Akt in cancer cells. (A and B) Down-regulation of XB130 decreased
phosphorylation of p21 and p27, and increased expression of p21 in WRO and A549 cells. Expressions of p27 and p53 were not affected by down-
regulation of XB130. (C and D) Down-regulation of XB130 decreased phosphorylations of FOXO3a and GSK3b in WRO and A549 cells. n=4. *p,0.05
(compared with control siRNA).
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org7 August 2012 | Volume 7 | Issue 8 | e43646
This may explain why both extrinsic and intrinsic signal induced
cell death was enhanced by XB130 siRNA treatment.
Phosphorylation of p21Cip1/WAF1 by Akt may result in its
cytoplasmic accumulation and promotes cell survival . Fork-
head family transcription factors can trigger apoptosis by inducing
the expression of genes that are critical for cell death, such as Bim
and Fas ligand [41,42]. Akt can phosphorylate FOXO3a, to block
its translocation from cytoplasm into the nucleus . Thus,
down-regulation of XB130 with siRNA reduced the phosphory-
lation of p21Cip1/WAF1 (Fig. 4A and B) and FOXO3a (Fig. 4C
and D). Taken together, these results suggest that XB130 is
involved in the regulation of cell proliferation and survival via
multiple molecules down-stream of PI3K/Akt pathway.
XB130 has been found to be a substrate and binding partner of
RET/PTC, an oncogenic protein tyrosine kinase . Recently,
we have found expression of XB130 in a variety of cell lines
derived from thyroid, lung, esophageal, pancreatic, and colon
cancers. In the present study, we demonstrated that XB130 is
involved in proliferation and survival of WRO thyroid cancer cells
(with RET/PTC mutation) and A549 lung carcinoma cells
(without RET/PTC mutation). Down-regulation of XB130 with
siRNA also reduced proliferation and survival of esophageal
cancer cells (data not shown). These results indicate that in
addition to RET/PTC XB130 may mediate activities of other
protein tyrosine kinases. Indeed, when co-expressed with XB130,
several receptor tyrosine kinases and non-receptor tyrosine kinases
were able to significantly increase phosphorylation of XB130. It
has been recently shown that Src family inhibitors, PP1 or PP2,
abolished cAMP-induced tyrosine phosphorylation of XB130 and
its interaction with p85 PI3K . Therefore, it is plausible that
XB130 could be a substrate of multiple protein tyrosine kinases,
and it may mediate cell-cycle progress and survival in multiple
cancer cell types.
In the last decade, there has been an increasing focus on the
PI3K/Akt pathway as one of the central pathways for cell
proliferation and survival [43,44,45]. The class-Ia PI3Ks are
heterodimers with a regulatory subunit (p85a, p85b or p55d) and
a p110 catalytic subunit (p110a, p110b or p110d) . Specific
phosphotyrosine residues on activated growth factor receptors or
on adaptor proteins can bind to SH2 domains of p85, which
increases the enzymatic activity of the p110 catalytic subunit and
also recruits the enzyme to membrane, where it catalyzes the
formation of lipid second messenger, PIP3, by phosphorylate
phosphoatidylinositol-4,5-bisphosphate (PIP2) at the 39 position on
its inositol ring  (Fig. 6). We have found a consensus p85
binding site (YxxM) in the N-terminus of XB130 that can bind to
either the N-or the C-terminal SH2 domain of p85a determined
by GST-fusion protein pull-down assays, and that XB130 is co-
immunoprecipitated with p85a in human thyroid carcinoma
TPC-1 cells. Phospho-peptide containing the YxxM sequence of
XB130 (but not its none-phosphorylated form) reduced XB130/
Figure 5. XB130 regulates cell survival through caspase-8 and caspase-9 signaling. (A and B) Cleaved fragments of both caspase-8 and-9
were distinctly increased by knocking-down of XB130 in WRO cells. In A549 cells, down-regulation of XB130 decreased procaspase-8 and increased
cleaved caspase-9. n=4. Mean 6 SEM. *p,0.05 (compared with control siRNA).
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org8 August 2012 | Volume 7 | Issue 8 | e43646
p85 interaction and deletion of N-terminus abolished interaction
between XB130 and p85 . In a recent study, Yamanaka et al.
found rat XB130 was associated with p85 subunit of PI3K, and
they named it as PI3KAP. This interaction is essential to enhance
cAMP-induced amplification of IGF mitogenic activity in FRTL-5
thyroid cells . In the present study, interaction between XB130
and p85 was found in both thyroid and lung cancer cells.
Importantly, down-regulation of XB130 affected many down-
stream proteins of PI3K/Akt pathway. These evidences indicate
that XB130 is an important regulator of PI3K pathway through its
specific binding with p85.
Activation of Akt is a feature of thyroid cancers [48,49,50].
Lung tumors also show a high level of phosphorylated Akt .
Treatment of thyroid or lung cancer cells with PI3K inhibitors,
LY294002 or wortmannin, resulted in decreased cell growth or
viability [48,49,50,52,53,54]. Due to its critical role in regulating
gene transcription, cell cycle progression, and survival, Akt has
been implicated in many types of cancer [47,55]. Identification of
XB130 as an effective up-stream regulator of PI3K/Akt pathway
may reveal new therapeutic targets for cancer therapeutics.
Oncoproteins may enhance tumor growth by altering cell cycle
progression and/or modulating cell death. Down regulation of
XB130 with siRNA not only reduced cell cycle progression but
also enhanced cell death, indicating that XB130 is a key upstream
regulator of both cellular processes. Our results indicate that
XB130 regulates cell cycle progression and survival through
multiple molecules, including
FOXO3a, GSK3b, Caspase 8 and Caspase 9 (Fig. 6). Although
we did not examine all known substrates of Akt, our results
strongly suggest that XB130 exerts its regulatory function of cell
proliferation and survival through the PI3K/Akt pathway.
Furthermore, down-regulation of XB130 did not affect down-
stream regulators FOXO1 and p53 indicating that in addition to
XB130 related Akt activity, these molecules can be further
regulated by other signaling mechanisms, which in turn ensures
specificity of XB130-related functions.
In summary, we showed that XB130 could regulate cell
proliferation and survival through modulating the PI3K/Akt
pathway in thyroid and lung cancer cells. XB130 could be a novel
oncoprotein in multiple cancers. RNA interference has become
a powerful tool to modulate gene function both in vitro and in vivo
[56,57]. Targeting XB130 expression with this technique could be
an attractive option for cancer therapy.
affect apoptosis in A549 cells in the presence of 10%
FBS. A549 cells were cultured in DMEB plus 10% FBS. (A)
Down-regulation of XB130 didn’t enhance spontaneous and
induced cell death. A549 cells were treated with 200 nM STS, or
500 ng/ml FasAb for 24 h. (B) FasAb (500 ng/ml) with IFN-c
(100 ng/ml) did not induce apoptosis, as analyzed by flow
cytometry using PI/Annexin V double staining. n=3. Mean 6
SEM. (C) Expression of Fas were confirmed in A549 and WRO
cells by western blotting.
Down-regulation of XB130 with siRNA did not
Figure 6. Roles of XB130 in cell cycle progression and survival of cancer. XB130 specifically binds p85a subunit of PI3K, which subsequently
activate Akt. Akt plays an essential role in cell proliferation and survival. Down-regulation of XB130 with siRNA affected multiple molecules down-
stream of Akt. This suggests that XB130 is an important regulator in PI3K/Akt related cancer cell proliferation and survival.
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org9 August 2012 | Volume 7 | Issue 8 | e43646
WRO and A549 cells transfected with control or XB130
siRNA. Phosphorylations of Src, ERK and JNK were not affected
by down-regulation of XB130 in WRO and A549 cells, whereas
phosphrylation of p38 was increase in WRO cells. n=4. Analyses
were performed by western blotting. Mean 6 SEM. *p,0.05
(compared with control siRNA).
Phosphorylation levels of Src and MAPKs in
Conceived and designed the experiments: AS ML. Performed the
experiments: AS XHB DI. Analyzed the data: AS GS ML. Contributed
reagents/materials/analysis tools: MS VD ML SK. Wrote the paper: AS
1. Shiozaki A, Liu M (2011) Roles of XB130, a novel adaptor protein, in cancer.
J Clin Bioinforma 1: 10.
2. Lodyga M, Bai XH, Mourgeon E, Han B, Keshavjee S, et al. (2002) Molecular
cloning of actin filament-associated protein: a putative adaptor in stretch-
induced Src activation. Am J Physiol Lung Cell Mol Physiol 283: L265–274.
3. Han B, Bai XH, Lodyga M, Xu J, Yang BB, et al. (2004) Conversion of
mechanical force into biochemical signaling. J Biol Chem 279: 54793–54801.
4. Xu J, Bai XH, Lodyga M, Han B, Xiao H, et al. (2007) XB130, a novel adaptor
protein for signal transduction. J Biol Chem 282: 16401–16412.
5. Lodyga M, Bai XH, Kapus A, Liu M (2010) Adaptor protein XB130 is a Rac-
controlled component of lamellipodia that regulates cell motility and invasion.
J Cell Sci 123: 4156–4169.
6. Santarpia L, Myers JN, Sherman SI, Trimarchi F, Clayman GL, et al. (2010)
Genetic alterations in the RAS/RAF/mitogen-activated protein kinase and
phosphatidylinositol 3-kinase/Akt signaling pathways in the follicular variant of
papillary thyroid carcinoma. Cancer 116: 2974–2983.
7. Dhanasekaran DN, Reddy EP (2008) JNK signaling in apoptosis. Oncogene 27:
8. Stiles BL (2009) PI-3-K and AKT: Onto the mitochondria. Adv Drug Deliv Rev
9. Tartari CJ, Donadoni C, Manieri E, Mologni L, Mina PD, et al. (2011)
Dissection of the RET/beta-catenin interaction in the TPC1 thyroid cancer cell
line. Am J Cancer Res 1: 716–725.
10. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, et al. (1990)
PTC is a novel rearranged form of the ret proto-oncogene and is frequently
detected in vivo in human thyroid papillary carcinomas. Cell 60: 557–563.
11. Kondo T, Ezzat S, Asa SL (2006) Pathogenetic mechanisms in thyroid follicular-
cell neoplasia. Nat Rev Cancer 6: 292–306.
12. Lodyga M, De Falco V, Bai XH, Kapus A, Melillo RM, et al. (2009) XB130,
a tissue-specific adaptor protein that couples the RET/PTC oncogenic kinase to
PI 3-kinase pathway. Oncogene 28: 937–949.
13. Balogh K, Asa SL, Zheng L, Cassol C, Cheng S, et al. (2011) The insulin
resistance Grb14 adaptor protein promotes thyroid cancer ret signaling and
progression. Oncogene: Epub ahead of print.
14. Shiozaki A, Lodyga M, Bai XH, Nadesalingam J, Oyaizu T, et al. (2011)
XB130, a novel adaptor protein, promotes thyroid tumor growth. Am J Pathol
15. Shiozaki A, Bai XH, Shen-Tu G, Moodley S, Takeshita H, et al. (2012) Claudin
1 Mediates TNFalpha-Induced Gene Expression and Cell Migration in Human
Lung Carcinoma Cells. PLoS One 7: e38049.
16. Tang PS, Tsang ME, Lodyga M, Bai XH, Miller A, et al. (2006)
Lipopolysaccharide accelerates caspase-independent but cathepsin B-dependent
death of human lung epithelial cells. J Cell Physiol 209: 457–467.
17. Iavarone C, Acunzo M, Carlomagno F, Catania A, Melillo RM, et al. (2006)
Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3,
a constitutively active form of the RET proto-oncogene. J Biol Chem 281:
18. Romano A, Wong WT, Santoro M, Wirth PJ, Thorgeirsson SS, et al. (1994) The
high transforming potency of erbB-2 and ret is associated with phosphorylation
of paxillin and a 23 kDa protein. Oncogene 9: 2923–2933.
19. Han B, Mura M, Andrade CF, Okutani D, Lodyga M, et al. (2005) TNFalpha-
induced long pentraxin PTX3 expression in human lung epithelial cells via JNK.
J Immunol 175: 8303–8311.
20. Mura M, Han B, Andrade CF, Seth R, Hwang D, et al. (2006) The early
responses of VEGF and its receptors during acute lung injury: implication of
VEGF in alveolar epithelial cell survival. Crit Care 10: R130.
21. Balogh K, Asa SL, Zheng L, Cassol C, Cheng S, et al. (2011) The insulin
resistance Grb14 adaptor protein promotes thyroid cancer ret signaling and
22. Noguchi PD, Johnson JB, Browne W (1981) Measurement of DNA synthesis by
flow cytometry. Cytometry 1: 390–393.
23. Tang PS, Mura M, Seth R, Liu M (2008) Acute lung injury and cell death: how
many ways can cells die? Am J Physiol Lung Cell Mol Physiol 294: L632–641.
24. Keane MM, Ettenberg SA, Lowrey GA, Russell EK, Lipkowitz S (1996) Fas
expression and function in normal and malignant breast cell lines. Cancer Res
25. Bernassola F, Scheuerpflug C, Herr I, Krammer PH, Debatin KM, et al. (1999)
Induction of apoptosis by IFNgamma in human neuroblastoma cell lines
through the CD95/CD95L autocrine circuit. Cell Death Differ 6: 652–660.
26. Fung SY, Oyaizu T, Yang H, Yuan Y, Han B, et al. (2011) The potential of
nanoscale combinations of self-assembling peptides and amino acids of the Src
tyrosine kinase inhibitor in acute lung injury therapy. Biomaterials 32: 4000–
27. Yamanaka D, Akama T, Fukushima T, Nedachi T, Kawasaki C, et al. (2012)
Phosphatidylinositol 3-kinase-binding protein, PI3KAP/XB130, is required for
cAMP-induced amplification of IGF mitogenic activity in FRTL-5 thyroid cells.
Mol Endocrinol 26: 1043–1055.
28. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, et al. (1996)
Mechanism of activation of protein kinase B by insulin and IGF-1. Embo J 15:
29. Viglietto G, Motti ML, Bruni P, Melillo RM, D’Alessio A, et al. (2002)
Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase
inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer.
Nat Med 8: 1136–1144.
30. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, et al. (2002) PKB/
Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-
mediated G1 arrest. Nat Med 8: 1153–1160.
31. Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, et al. (2002) PKB/Akt mediates
cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and
modulation of its cellular localization. Nat Med 8: 1145–1152.
32. Gartel AL, Radhakrishnan SK (2005) Lost in transcription: p21 repression,
mechanisms, and consequences. Cancer Res 65: 3980–3985.
33. Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, et al. (2001) Cytoplasmic
localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-
overexpressing cells. Nat Cell Biol 3: 245–252.
34. Seoane J, Le HV, Shen L, Anderson SA, Massague J (2004) Integration of Smad
and forkhead pathways in the control of neuroepithelial and glioblastoma cell
proliferation. Cell 117: 211–223.
35. Medema RH, Kops GJ, Bos JL, Burgering BM (2000) AFX-like Forkhead
transcription factors mediate cell-cycle regulation by Ras and PKB through
p27kip1. Nature 404: 782–787.
36. Cohen P, Frame S (2001) The renaissance of GSK3. Nat Rev Mol Cell Biol 2:
37. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, et al. (2001) HER-2/neu induces
p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol 3:
38. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, et al. (1998)
Regulation of cell death protease caspase-9 by phosphorylation. Science 282:
39. Rokhlin OW, Guseva NV, Tagiyev AF, Glover RA, Cohen MB (2002) Caspase-
8 activation is necessary but not sufficient for tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)-mediated apoptosis in the prostatic carcino-
ma cell line LNCaP. Prostate 52: 1–11.
40. Ping B, He X, Xia W, Lee DF, Wei Y, et al. (2006) Cytoplasmic expression of
p21CIP1/WAF1 is correlated with IKKbeta overexpression in human breast
cancers. Int J Oncol 29: 1103–1110.
41. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, et al. (1999) Akt promotes cell
survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell
42. Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, et al. (1999)
Direct control of the Forkhead transcription factor AFX by protein kinase B.
Nature 398: 630–634.
43. Franke TF, Kaplan DR, Cantley LC (1997) PI3K: downstream AKTion blocks
apoptosis. Cell 88: 435–437.
44. Liang J, Slingerland JM (2003) Multiple roles of the PI3K/PKB (Akt) pathway in
cell cycle progression. Cell Cycle 2: 339–345.
45. Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream.
Cell 129: 1261–1274.
46. Okkenhaug K, Vanhaesebroeck B (2003) PI3K in lymphocyte development,
differentiation and activation. Nat Rev Immunol 3: 317–330.
47. Luo J, Manning BD, Cantley LC (2003) Targeting the PI3K-Akt pathway in
human cancer: rationale and promise. Cancer Cell 4: 257–262.
48. Ringel MD, Hayre N, Saito J, Saunier B, Schuppert F, et al. (2001)
Overexpression and overactivation of Akt in thyroid carcinoma. Cancer Res
49. Liu W, Asa SL, Ezzat S (2005) 1alpha,25-Dihydroxyvitamin D3 targets PTEN-
dependent fibronectin expression to restore thyroid cancer cell adhesiveness.
Mol Endocrinol 19: 2349–2357.
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org 10August 2012 | Volume 7 | Issue 8 | e43646
50. Liu W, Asa SL, Fantus IG, Walfish PG, Ezzat S (2002) Vitamin D arrests thyroid Download full-text
carcinoma cell growth and induces p27 dephosphorylation and accumulation
through PTEN/akt-dependent and-independent pathways. Am J Pathol 160:
51. Massion PP, Taflan PM, Shyr Y, Rahman SM, Yildiz P, et al. (2004) Early
involvement of the phosphatidylinositol 3-kinase/Akt pathway in lung cancer
progression. Am J Respir Crit Care Med 170: 1088–1094.
52. Moore SM, Rintoul RC, Walker TR, Chilvers ER, Haslett C, et al. (1998) The
presence of a constitutively active phosphoinositide 3-kinase in small cell lung
cancer cells mediates anchorage-independent proliferation via a protein kinase B
and p70s6k-dependent pathway. Cancer Res 58: 5239–5247.
53. Brognard J, Clark AS, Ni Y, Dennis PA (2001) Akt/protein kinase B is
constitutively active in non-small cell lung cancer cells and promotes cellular
survival and resistance to chemotherapy and radiation. Cancer Res 61: 3986–
54. Razzini G, Berrie CP, Vignati S, Broggini M, Mascetta G, et al. (2000) Novel
functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in
human cancer cell lines. Faseb J 14: 1179–1187.
55. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway
in human cancer. Nat Rev Cancer 2: 489–501.
56. Behlke MA (2006) Progress towards in vivo use of siRNAs. Mol Ther 13: 644–
57. Akhtar S, Benter IF (2007) Nonviral delivery of synthetic siRNAs in vivo. J Clin
Invest 117: 3623–3632.
Roles of XB130 in Cancer
PLOS ONE | www.plosone.org11August 2012 | Volume 7 | Issue 8 | e43646