XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell
Huang et al. Journal of Hematology & Oncology
XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage- independent growth of bladder cancer cell
Chao Huang 0
Xingruo Zeng 0
Guosong Jiang 0
Xin Liao 0
Claire Liu 0
Jingxia Li 0
Honglei Jin 0
Junlan Zhu 0
Hong Sun 0
Chuanshu Huang 0
0 Nelson Institute of Environmental Medicine, New York University School of Medicine , 57 Old Forge Road, Tuxedo, NY 10987 , USA
Background: The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. Anti-IAP therapy is reported to be closely related to epidermal growth factor receptor (EGFR) expression level. However, whether and how XIAP modulates EGFR expression remains largely unknown. Methods: Human XIAP was knockdown with short-hairpin RNA in two different bladder cancer cell lines, T24T and UMUC3. Two XIAP mutants, XIAP ΔBIR (deletion of N-terminal three BIR domains) and XIAP ΔRING (deletion of C-terminal RING domain and keeping the function of BIR domains), were generated to determine which domain is involved in regulating EGFR. Results: We found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficiency of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the protein phosphatase 2 (PP2A) activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a. Conclusions: Our study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy.
XIAP BIR domain; miRNA; EGFR; Rac1; Bladder cancer
The X-linked inhibitor of apoptosis protein (XIAP) is a
member of the inhibitors of apoptosis (IAP) family, which
mainly function as suppressors of apoptosis. XIAP is
aberrantly increased in a variety of human cancers, including
acute leukemia , ovarian carcinoma , bladder cancer
, clear cell renal cancer [4, 5], and many other cancers
[6–8]. Elevated expression of XIAP mediates the
resistance of cancer cells to chemotherapeutic drugs, as well as
radiotherapy [9, 10]. Downregulation of XIAP has been
shown to sensitize drug-resistant cancer cells to
chemotherapeutic agents-induced apoptosis [11–13]. Therefore,
as an important diagnostic and prognostic biomarker in
many cancers [3, 14], XIAP may serve as a potential
therapeutic target for antineoplastic therapy.
XIAP contains three baculovirus IAP repeat domains
(BIR 1-3) in the N-terminal of the protein and one RING
domain in the C-terminal. In which, BIR1 interacts with
proteins that modulate NF-kappaB signaling  and BIR2
and BIR3 are critical for the interaction with either
caspase3 or -7 (BIR2) or caspase-9 (BIR3), respectively. The RING
domain can function as an E3 ligase and mediate the
proteasomal degradation of itself and bind to proteins such as
caspase-3 or the mitochondrial XIAP-inhibitor
SMAC/Diablo [16, 17]. Other than inhibiting apoptosis, XIAP is
involved in many cellular functions related to the cancer
malignancy. Our recent studies show that XIAP upregulates
cyclin D1 via its C-terminal RING domain to promote
bladder cancer cell growth  and enhances colorectal cancer
cell motilities through inhibiting the RhoGDIα SUMOlation
at the lys-138 site [19, 20]. Moreover, our recent study
reveals that XIAP directly binds to E2F1 via N-terminal BIR
domains and enhances E2F1 transcriptional activity, which
subsequently promotes colorectal cancer cell growth .
In addition, inhibition of XIAP activity by Embelin in
bladder cancer cells not only reduces cell viability but also
affects cell invasion in vitro, suggesting an important role of
XIAP in tumor formation and progression .
The epidermal growth factor receptor (EGFR) is a protein
mainly existing in the cell membrane and can be activated
by binding of its specific ligands, including epidermal
growth factor (EGF) and transforming growth factor α
(TGFα). EGFR overexpression has been widely reported in
a number of cancers, including breast, ovarian, bladder,
non-small-cell lung (NSCLC), colorectal cancers, and
other tumors [23–29]. Aberrant activation or expression
of EGFR leads to the promotion of proliferation, cell
motility, and invasive capacity of tumor [30–33]. As a
survival signaling protein, EGFR is widely used as a
therapeutic target. Many anti-EGFR-based therapeutic
drugs have been developed, such as monoclonal
antibodies cetuximab and panitumumab and tyrosine kinase
inhibitors like gefitinib and afatinib , which exhibit the
improved therapeutic effect in the patients.
A recent study shows that tumor cells overexpressing
EGFR are more sensitive to anti-IAPs therapy ,
suggesting a potential relationship between XIAP and
EGFR in cancer cells. However, whether and how XIAP
modulates EGFR expression remains largely unknown.
In the current study, we found that XIAP BIR domain
could regulate EGFR expression through
downregulating miR-200a by targeting protein phosphatase 2 (PP2A)/
c-Jun axis and further promoted the bladder cancer cell
Cell culture and reagents
UMUC3 cells and 293 T cells were cultured in DMEM
medium (Invitrogen, Carlsbad, CA, USA) supplemented
with 10% FBS, and T24T cells were cultured in
RPIM1640/F12 medium (Invitrogen, Carlsbad, CA, USA)
with 5% FBS. All cells were maintained in a humidified
incubator at 37 °C, with a 5% CO2 atmosphere. XIAP
polyclonal antibody was purchased from BD PharMingen (San
Diego, CA, USA); Antibodies specific against EGFR,
STAT3, p52, p65, p50, RelB, c-Jun, p38, Erk1/2, MKK,
MEK1/2, and PP2A were bought from Cell Signaling
Technology Inc. (Beverly, MA, USA); The specific
antibodies against Sp1, E2F1, β-actin and α-Tubulin were
from Santa Cruz Biotechnology (Santa Cruz, CA, USA);
the antibodies for GAPDH and JNK1/2 were bought from
GeneTex, Inc. (Irvine, CA, USA). Okadaic acid was
purchased from Santa Cruz Biotechnology (Santa Cruz,
Plasmids and stable cell transfection
The short-hairpin RNA (shRNA)-specific targeting human
XIAP was purchased from Open Biosystems (Lafayette,
CO, USA). The overexpression of miR-200a/200b/429
expression plasmid was bought from Addgene (Cambridge,
MA, USA). The miR-200a knockdown plasmid was
purchased from GeneCopoeia (Rockville, MD, USA). The
overexpression of HA-ΔRING plasmid, the TAM67
plasmid, a well-characterized dominant-negative c-Jun
mutant, and the p100 overexpression plasmid were described
in our previously studies [19, 36, 37]. Wild-type EGFR
3′UTR luciferase reporter is a kind gift from Professor
Benjamin Purow (University of Virginia Health System, VA)
. The miR-200a binding site point mutation of EGFR
3′-UTR luciferase reporter was constructed in our lab
based on the wild-type EGFR 3′-UTR luciferase reporter.
Cell transfections were performed with PolyJet™ DNA
In Vitro Transfection Reagent (SignaGen Laboratories,
Rockville, MD, USA) according to the manufacturer’s
instructions. For stable transfection, cell cultures were
subjected to hygromycin B, G418, or puromycin
selection according to the resistance of plasmids, and cells
surviving were pooled as stable mass transfectants. The
miR200a inhibitor lentivirus was packaged in 293T cells
with pCMV delta R8.2 and pMD2.G as described in the
previous publication .
Anchorage-independent growth assay
Anchorage-independent growth ability was evaluated in
soft agar as described in our previous studies .
Briefly, 3 ml of 0.5% agar in basal modified Eagle’s
medium supplemented with 10% FBS was layered onto
each well of 6-well tissue culture plates; 1 ml of 0.35%
agar medium with cells (1 × 104 cells) was added to each
well on top of the concretionary 0.5% agar layer. Plates
were incubated at 37 °C in 5% CO2 for 2–3 weeks, and
the colonies with more than 32 cells were scored and
are presented as colonies/104 cells.
Western blot analysis
Whole cell extracts were prepared with the cell lysis
buffer (10 mM Tris-HCl, pH 7.4, 1% SDS, and 1 mM
Na3VO4) as described in our previous studies ; 50 μg
of proteins were resolved by SDS-PAGE, transferred to a
PVDF membrane, and probed with the indicated primary
antibodies together with the AP-conjugated secondary
antibody. Signals were detected by the enhanced
chemifluorescence Western blotting system as described in the
previous report . The images were acquired by
scanning with the phosphorimager (Typhoon FLA 7000
imager; Pittsburgh, PA, USA).
Luciferase reporter assay
EGFR mRNA 3′ UTR luciferase reporter, miR-200a/200b/
429 promoter luciferase reporter, was stably transfected
into cultured cells. Luciferase activity was determined by
using the luciferase Assay System kit (Promega, Madison,
WI, USA). The results were normalized by internal TK
signal. All experiments were done in triplicates and the
results expressed as mean ± standard error (SE).
Quantitative real-time PCR
For mRNA detection: total RNA was extracted using the
TRIzol reagent as described in the manufacturer’s
instructions (Invitrogen, Grand Island, NY, USA); 5 μg
total RNA was used for first-strand cDNA synthesis with
oligdT primer by SuperScript IV First-Strand Synthesis
system (Invitrogen, Grand Island, NY, USA). The PCR
was done using PowerUp SYBR Green Master Mix
(Invitrogen, Grand Island, NY, USA) specifically. The primers
used in this study were human egfr, forward: 5′-CCA
AGG CAC GAG TAA CAA GC-3′, reverse: 5′-AGG
GCA ATG AGG ACA TAA CCA G-3′; and human
gapdh, forward: 5′-AGA AGG CTG GGG CTC ATT
TG-3′, reverse: 5′-AGG GGC CAT CCA CAG TCT
TC-3′. The initial activation was performed at 50 °C for
2 min, 95 °C for 10 min, and followed by 40 cycles (95 °C
for 15 s, 60 °C for 1 min).
For miRNA detection: cells used for total RNA
extraction using miRNeasy Mini Kit (QIAGEN, Valencia, CA,
USA); 1 μg total RNA was used for reverse transcription.
Analysis of miRNAs was done using miScript PCR system
(QIAGEN, Valencia, CA, USA) by QuantStudio Real-time
PCR system (Applied Biosystems). The primers were
purchased from Invitrogen (Grand Island, NY, USA), and U6
was used for inner control. The initial activation was
performed at 95 °C for 15 min and followed by 40 cycles,
denaturation at 95 °C for 15 s, annealing at 55 °C for 30s,
and extension at 70 °C for 30s.
Student’s T test was used to determine the significance
between different groups. p < 0.05 was considered as a
significant difference between compared groups.
BIR domain is required for XIAP-mediated EGFR protein
expression and anchorage-independent growth in
bladder cancer cells
To investigate the functional interplay between XIAP
and EGFR, we first examined whether XIAP affected
EGFR expression levels in human bladder cancer cells. A
short-hairpin RNA (shRNA) specifically targeting human
XIAP was used to knockdown endogenous XIAP in two
different bladder cancer cell lines, T24T and UMUC3.
The stable transfectants of control shRNA (Nonsense)
and XIAP shRNA (shXIAP) were established and analyzed
for EGFR expression. As shown in Fig. 1a, knockdown of
XIAP in both cell lines resulted in a significant reduction
of EGFR levels, suggesting a crucial role of XIAP in EGFR
expression. Similar to our previous finding in XIAP−/−
HCT116 cells , depletion of XIAP in T24T and
UMUC3 cells (shXIAP) resulted in a marked reduction of
anchorage-independent growth (Fig. 1c, d). Interestingly,
ectopically expressing EGFR in XIAP knockdown cells
completely recovered the number of colonies that grew in
soft agar, indicating that reduced EGFR expression in
shXIAP cells indeed contribute to the reduced
anchorageindependent growth capacity of these cells (Fig. 1e–g).
XIAP contains N-terminal BIR domains and C-terminal
RING domain. To identify which domain is involved in
regulating EGFR expression, two XIAP mutants were
generated: XIAP ΔBIR (deletion of N-terminal three BIR
domains) and XIAP ΔRING (deletion of C-terminal RING
domain and keeping the function of BIR domains) .
The stable transfectants of ΔRING and ΔBIR in XIAP
knockdown cells were established (Fig. 1a, b).
Interestingly, while cells overexpressing XIAP ΔRING resumed
Fig. 1 BIR domain is required for XIAP-mediated EGFR protein expression and anchorage-independent growth in bladder cancer cells. a, b The
cell extracts obtained from stable transfectants, T24T(Nonsense), T24T(shXIAP/Vector), T24T(shXIAP/ΔRING), UMUC3(Nonsense), UMUC3(shXIAP/
Vector), UMUC3(shXIAP/ΔRING), UMUC3(shXIAP/Vector), or UMUC3(shXIAP/ΔBIR), were subjected to Western blot for determination of expression
of XIAP and EGFR. GAPDH was used as the protein loading control. c, d The indicated stable transfectants were used for determination of their
anchorage-independent growth ability in soft agar assay. Colonies with more than 32 cells were scored and presented as colonies/104 cells.
Results were presented as means ± SD from triplicates. The asterisk “*” indicates a significant decrease as compared with nonsense transfectant,
while symbol “※” indicates a significant increase in comparison to scramble vector transfectant (p < 0.05). Error bars represent S.D. e The cell
extracts from T24T(shXIAP/Vector), T24T(shXIAP/EGFR-GFP), UMUC3(shXIAP/Vector), and UMUC3(shXIAP/EGFR-GFP), were subjected to Western
blot for determination of indicated protein expression. β-actin was used as the protein loading control. f, g The indicated stable transfectants
were used for determination of their anchorage-independent growth ability in soft agar assay. Colonies with more than 32 cells were scored and
presented as colonies/104 cells. Results were presented as means ± SD from three independent experiments. The asterisk “*” indicates a significant
increase as compared with the scramble vector transfectant (p < 0.05). Error bars represent S.D.
EGFR protein levels in XIAP knockdown cells, the cells
overexpressing ΔBIR retained low EGFR levels similar to
vector control transfectant (empty vector) (Fig. 1a, b),
suggesting that BIR domain, not RING domain, plays a
critical role in regulating EGFR expression. Moreover,
overexpressing ΔRING not only resumed the EGFR
levels but also rescued anchorage-independent growth
(Fig. 1c, d). These results indicate that BIR domain is
required for XIAP-dependent EGFR expression and
XIAP BIR domain regulated the EGFR expression at
translation level through increasing EGFR mRNA 3′ UTR
Since overexpressing XIAP ΔBIR did not affect the
EGFR expression, only XIAP ΔRING mutant was used
to elucidate the molecular mechanisms underlying the
XIAP regulation of EGFR expression. We first analyzed
the mRNA level of EGFR in both T24T and UMUC3
stable transfectants expressing nonsense,
shXIAP/Vector, and shXIAP/ΔRING. As shown in Fig. 2a, b, no
significant change of EGFR mRNA in all three
transfectants, suggesting that XIAP-mediated regulation
of EGFR expression was beyond mRNA level. To
determine whether XIAP regulates EGFR protein via
proteasome-mediated degradation, T24T cells expressing
nonsense, shXIAP/Vector, and shXIAP/ΔRING, were
pretreated with or without MG132, a proteasome
inhibitor, for 6 h as indicated to accumulate the EGFR protein.
Cycloheximide (CHX) was then used at different time
periods to observe EGFR degradation rates among the
cell transfectants. The results showed that there was no
observable difference between the three established cells
(Fig. 2c), indicating that the difference of EGFR
expression is not regulated by the proteasome-mediated
protein degradation. Next, we examined the potential
contribution of untranslated region (UTR) in the
regulation of EGFR expression. As shown in Fig. 2d, EGFR 3′
UTR activity was inhibited in XIAP knockdown cells
and overexpression of the ΔRING reversed this
inhibition. Thus, XIAP BIR domain may regulate EGFR at
translation level through increasing EGFR 3′ UTR
Fig. 2 XIAP BIR domain regulated the EGFR expression at translation level through increasing EGFR mRNA 3′ UTR activity. a, b The EGFR mRNA
expression level was evaluated by real-time PCR in both T24T and UMUC3 cells, and GAPDH mRNA was used as the internal loading control.
Results were presented as the mean ± SD from triplicates. c The indicated stable transfectants were pre-treated with or without MG132 (10 μM)
for 6 h and then treated with cycloheximide (CHX, 100 μg/ml) as indicated time interval. The cell extracts were subjected to Western blotting for
determination of EGFR degradation, α-Tubulin was used as the protein loading control. d EGFR 3′ UTR luciferase reporter was stably transfected
into cells as indicated, and luciferase activity was evaluated by the Dual-Luciferase Reporter Assay System. Results were presented as the mean ± SD
from triplicates. The asterisk “*” indicates a significant inhibition as compared with nonsense transfectant, while symbol “※” indicates a significant
increase in comparison to vector transfectant (p < 0.05). Error bars represent S.D.
XIAP BIR domain-mediated inhibition of miR-200a was
responsible for EGFR protein translational regulation by
targeting EGFR mRNA 3′ UTR
microRNA, a class of ~22-nucletide noncoding small
RNAs, had been reported to bind to the 3′ UTR of its
targeted genes and inhibit their protein translation .
By using online prediction tools Targetscan 6.2 and
starBase v2.0 [44, 45], we obtained a number of miRNAs
that potentially target EGFR 3′ UTR, including miR-7,
miR-27a, miR-27b, miR-133a, miR-133b, miR-141,
miR200a, and miR-302b. To determine specific miRNAs that
mediate XIAP-dependent EGFR expression, we analyzed
the levels of these miRNAs in T24T(Nonsense) and
T24T(shXIAP) cells (Fig. 3a). The results obtained from
quantitative real-time PCR indicated that only miR200a
was significantly increased upon knockdown of XIAP,
indicating that miR-200a is the potential miRNA that
might be negatively regulated by XIAP. To further test
this notion, the levels of the miR200a-200b-429 cluster
were analyzed in T24T(Nonsense), T24T(shXIAP), and
T24T(shXIAP/ΔRING) cells. As shown in Fig. 3b, the
upregulation of miR-200a by knockdown of XIAP was
impaired by overexpression of BIR domain, while
alteration of miR-200b and miR-429 was not observed by
Fig. 3 BIR domain-mediated inhibition of miR-200a was responsible for XIAP promotion of EGFR protein translational regulation by targeting EGFR
mRNA 3′ UTR. a The indicated microRNAs, which could potential bind to EGFR mRNA 3′ UTR, were evaluated by real-time PCR. Results were presented
as the mean ± SD from triplicates. The asterisk “*” indicates a significant increase as compared with nonsense transfectant (p < 0.05). b Expression levels
of the miR-200a/200b/429 cluster in the indicated three stable cells were evaluated by real-time PCR. Results were presented as the mean ± SD of
triplicates. The asterisk “*” indicates a significant change as compared with nonsense transfectant (p < 0.05), while symbol “※” indicates a significant
decrease in comparison to vector transfectants (p < 0.05). c miR-200a expression plasmid was stably transfected to T24T cells and the expression
efficiency was determined by real-time PCR. Results were presented as the mean ± SD of triplicates. The asterisk “*” indicates a significant increase as
compared to that in T24T(Vector) (p < 0.05). d T24T(Vector) and T24T(miR-200a) cells were subjected to Western blotting to determine the
EGFR expression. GAPDH was used as the protein loading control. e miR-200a inhibitor lentivirus was used to infect T24T(shXIAP) cells and the
knockdown efficiency was determined by real-time PCR. Results were presented as the mean ± SD of triplicates. The asterisk “*” indicates a
significant decrease as compared to control (p < 0.05). f The indicated cells were subjected to Western blotting to determine the EGFR expression.
GAPDH was used as the protein loading control. g EGFR 3′-UTR Luciferase reporter and its miR-200a binding site point mutation were diagramed as
indicated. h Wild-type (WT) and mutated (Mut) of EGFR 3′-UTR Luciferase reporters were stably co-transfected with miR-200a or its scramble vector,
and the stable transfectants were used to evaluate for their reporter activity. Results were presented as the mean ± SD of triplicates. The asterisk “*”
indicates a significant inhibition as compared with vector transfectant (p < 0.05). Error bars represent S.D.
either knockdown of XIAP or overexpression of BIR
domain, revealing the important role of the BIR domain in
XIAP inhibition of miR-200a expression. To evaluate the
effect of miR-200a on EGFR, we generated stable T24T
transfectant expressing miR-200a, which showed over
22-fold expression of miR-200a in comparison to the
corresponding empty vector transfectant (Fig. 3c).
Ectopic expression of miR-200a in T24T cells led to a
dramatical reduction of EGFR expression (Fig. 3d), and
inhibited expression of miR-200a in T24T(shXIAP) cells
could also significantly upregulate the EGFR expression
(Fig. 3e, f ). These results suggested that EGFR mRNA
was likely to be targeted by miR-200a. To clarify if
miR200a could directly bind to 3′ UTR of the EGFR mRNA,
we introduced point mutations of miR-200a binding site
in EGFR 3′ UTR as indicated in Fig. 3g. The results
indicated that overexpressed miR-200a failed to inhibit the
mutant EGFR 3′ UTR activity, whereas it significantly
inhibited the activity in wild-type EGFR 3′ UTR reporter
(Fig. 3h). These results demonstrate that the BIR domain
of XIAP promotes EGFR protein expression through
suppression of miR-200a expression.
XIAP BIR domain promoted miR-200a transcription by
inhibiting c-Jun protein phosphorylation at Ser63/73
To elucidate the molecular mechanisms underlying XIAP
regulation of miR-200a, the promoter activity of miR-200a
was evaluated and compared among T24T(Nonsense),
T24T(shXIAP/Vector), and T24T(shXIAP/ΔRING) cells.
As shown in Fig. 4a, knockdown of XIAP significantly
increased the promoter activity of miR-200a, overexpressing
ΔRING in XIAP knockdown cells reversed the miR-200a
promoter activity in the T24T(shXIAP/ΔRING) cells. We
next performed a bioinformatics scan on the promoter
region of miR-200a, and several potential binding sites for
transcription factors were shown in the miR-200a
promoter region, including the binding sites for c-Jun, E2F1,
STAT3, NF-κB, and Sp-1 (Fig. 4b). To define the specific
transcription factors involved in the regulation of
miR200a, we determined the expression of these transcription
Fig. 4 XIAP BIR domain promoted miR-200a transcription by inhibiting c-Jun protein phosphorylation at Ser63/73. a miR-200a promoter luciferase
activity was evaluated by the Dual-Luciferase Reporter Assay System. Results are the mean ± SD of triplicates. The asterisk “*” indicates a significant
increase as compared with nonsense cells (p < 0.05). The symbol “※” indicates a significant inhibition as compared with vector transfectant (p < 0.05).
b The diagram of predicted transcription factor binding sites in miR-200a promoter region. c Western blotting was used to analyze the transcription
factors expression, and GAPDH was used as the protein loading control. d Short hairpin RNA-specific targeting human RelB were stably transfected to
UMUC3(shXIAP) cells, and Western blotting was used to determine the knockdown efficiency and EGFR expression, while GAPDH was used as the
protein loading control. e p100 was transiently transfected to T24T cells, and Western blotting was used to determine the expression of p100 and
EGFR, and β-actin was used as the protein loading control. f TAM67 was stably transfected into UMUC3 cells, and real-time PCR was used to determine
the miR-200a expression. Results were presented as the mean ± SD of triplicates. The asterisk “*” indicates a significant inhibition as compared with
vector transfectants (p < 0.05). g The cell extracts from UMUC3(Vector) and UMUC3(TAM67) were subjected to Western blot for determination of the
indicated protein expression, and GAPDH was used as the protein loading control
factors in various stable transfectants of T24T and
UMUC3 cell lines as indicated in Fig. 4c. Among these
transcription factors tested, the levels of RelB, p100
protein, and c-Jun phosphorylation (Ser63/73) were
upregulated in XIAP knockdown cells, and this upregulation was
reversed by ectopic expression of ΔRING, suggesting they
are consistent with alteration of miR-200a in those
transfectants (Fig. 4c). Therefore, we knocked down RelB in
UMUC3(shXIAP) cells and its effect on EGFR expression
was evaluated. The results showed that knockdown of
RelB exhibited little effect on EGFR expression (Fig. 4d).
As a precursor of p52, p100 could function in
p52independent fashion . Thus, we next evaluated the
potential contribution of p100 to EGFR expression by
ectopic expressing p100 in T24T cells. As shown in Fig. 4e,
overexpression of p100 resulted in an upregulation of
EGFR, which was inconsistent with the regulatory effect
of XIAP on EGFR, excluding p100 participating in XIAP
regulation of EGFR expression. Further, c-Jun
dominantnegative mutant expression plasmid TAM67 was
transfected into UMUC3 cells for determining the potential
contribution of c-Jun activation to the expression of
miR200a and EGFR. As expected, ectopic expression of
dominant-negative c-Jun (protein product named as
cJun(D)), successfully blocked miR-200a expression (Fig. 4f )
and increased EGFR protein levels (Fig. 4g),
demonstrating an important role of c-Jun activation in XIAP
suppression of miR-200a and upregulation of EGFR expression.
Taken together, our results demonstrate that BIR domain
mediates XIAP inhibition of c-Jun activation (Ser63/73
phosphorylation) and subsequently suppresses miR-200a
expression and promotes EGFR protein translation.
PP2A/MAPKK/MAPK axis was a BIR domain downstream
effector mediated inhibition of c-Jun protein
Phosphorylation of c-Jun at Ser63/73 is regulated by all
three MAP kinases, including JNK1/2, p38, and Erk1/2.
To determine which mitogen-activated protein kinase
(MAPK) has involved in XIAP-mediated c-Jun
inactivation, we analyzed the levels and activation of these
MAPKs in UMUC3(Nonsense),
UMUC3(shXIAP/vector), and UMUC3(shXIAP/ΔRING). Surprisingly, all
three MAPKs were activated in XIAP knockdown cells
(Fig. 5a), while overexpression of ΔRING impaired those
activations (Fig. 5a). Those results revealed a crucial role
of BIR domain in XIAP inhibition of MAPK pathways,
which led us to further examine the MAPK upstream
kinases and phosphatases. As shown in Fig. 5b, almost all
MAPK kinase except MKK7 exhibited the similar
increases in the phosphorylation, suggesting that, instead
of targeting each individual MAPK kinase (MAPKK)/
MAPK, XIAP and its BIR domain might elicit a much
broader effect across all MAPKK/MAPK pathways. Our
previous studies have demonstrated that protein
phosphatase 2 (PP2A), a major serine-threonine phosphatase
that counteracts with activation of MAPKK/MAPK
pathway , is able to regulate the c-Jun phosphorylation
. To test if PP2A is involved in this XIAP-regulated
MAPK/c-Jun activation, we evaluated various units of
PP2A protein levels in UMUC3(Nonsense),
UMUC3(shXIAP/Vector), and UMUC3(shXIAP/ΔRING) cells. As
shown in Fig. 5c, phosphorylation of PP2A-C subunit at
Tyr307 (inactivate form of PP2A) was remarkably
increased in UMUC3(shXIAP/Vector) cells, but returned to
the similar level observed in UMUC3(Nonsense) cells
when overexpressing ΔRING domain, while other
subunits, including PP2A-A and PP2A-B, did not show the
consistent alteration. These results suggest that XIAP and
its BIR domain provide an inhibitory effect on PP2A-C
subunit phosphorylation at Tyr307 in bladder cancer cells.
These results indicate that alteration of PP2A
phosphorylation at Tyr307 may involve in XIAP/BIR regulation of
the MAPK/c-Jun phosphorylation.
To test whether inactivation of PP2A in
UMUC3(shXIAP) cells mediates activation of
MAPKKs/MAPKs/cJun, in turn reducing EGFR expression, we treated
UMUC3(shXIAP/Vector) and UMUC3(shXIAP/ΔRING)
cells with Okadaic acid (OA), which is a specific PP2A
and protein phosphatase 1 (PP1) inhibitor , and the
activation of MAPKKs/MAPKs/c-Jun, as well as EGFR
expression, were evaluated. As shown in Fig. 5d, the
treatment of cells with OA led to similar elevations of PP2A-C
phosphorylation at Tyr307 accompanied by enhanced
activation of MAPKKs/MAPKs/c-Jun as well as decreased
EGFR expression in both UMUC3(shXIAP/Vector) and
UMUC3(shXIAP/ΔRING) cells. These results indicate
that the inactivation of PP2A-C by elevation of
phosphorylation at Tyr307 mediates activation of MAPK/
MAPKKs/c-Jun, induction of miR-200a, as well as
inhibition of EGFR expression in UMUC3(shXIAP/Vector)
cells, further supporting that XIAP/BIR-regulated PP2A
plays an important role in their mediating EGFR
XIAP BIR domain inhibited Rac1 expression and
subsequently resulted in the downregulation of PP2A-C
Phosphorylation at Tyr307
The previous study identified that Rac1 could binding
to SET, also known as TAF1β (template activating
factor 1β), and suppress the function of PP2A by
increasing the phosphorylation level in tumor cells [48, 49].
Also, it has been reported that in cardiac myocytes,
Rac1/CDC42 complex promotes PP2A activity by
dephosphorylating PP2A through upregulating p21-activated
kinase-1 (Pak1) activity . To determine whether Rac1
and CDC42 were involved in the BIR regulation of
PP2A, we evaluated the expression of Rac1 and CDC42
Fig. 5 PP2A/MAPKK/MAPK axis was a BIR domain downstream effector responsible for its inhibition of c-Jun protein phosphorylation and activation.
a–c Whole cells lysis obtained from the indicated transfectants were subjected to Western blot for analysis of activation of the MAPKs (a) and MAPKKs
(b) and expression of PP2A. GAPDH was used as the protein loading control. d The indicated cells were treated with Okadaic acid (OA) for 6 h, and
whole cells lyses were subjected to Western blot for determination of protein expression. GAPDH was used as the protein loading control
in UMUC3(Nonsense), UMUC3(shXIAP/Vector), and
UMUC3(shXIAP/ΔRING) cells. The results showed
that knockdown of XIAP remarkably increased the
Rac1 expression and the introduction of ΔRING
reversed the Rac1 expression (Fig. 6a). In contrast to
Rac1, CDC42 showed the opposite alterations (Fig. 6a),
which minimized the possibility of Rac1/CDC42
complex participating in XIAP regulation of PP2A activity
and further suggesting that Rac1 might contribute to
this regulation. As expectedly, overexpression of Rac1
in UMUC3(shXIAP/ΔRING) cells led to
downregulation of EGFR and upregulation of the PP2A-C
phosphorylation at Tyr307 as well as c-Jun phosphorylation
at Ser63/Ser73 (Fig. 6b). Consistently, the 3′ UTR
activity of EGFR was significantly downregulated in the
same cells (Fig. 6c). These data demonstrate that XIAP and
its BIR domain inhibit Rac1 expression and subsequently
decreasing the PP2A-C phosphorylation at Tyr307 and
MAPKK/MAPK/c-Jun activation, and further resulting in
miR-200a induction and EGFR translation inhibition, as
diagramed in Fig. 6d.
Although recent studies show the co-overexpression of
XIAP and EGFR in many cancers [14, 35, 51, 52], and the
high sensitive to anti-IAPs therapy if the cancers with
Fig. 6 XIAP BIR domain inhibited Rac1 expression and subsequently resulted in the downregulation of PP2A-C Phosphorylation at Tyr307. a, b The
whole extracts were subjected to Western blot for determination of protein expression as the indicated. GAPDH was used as the protein loading
control. c EGFR mRNA 3′ UTR-luciferase reporter was stably transfected to UMUC3(shXIAP/ΔRING), and the luciferase activity was evaluated by the
Dual-Luciferase Reporter Assay System. Results were presented as the mean ± SD of triplicates. The asterisk “*” indicates a significant inhibition as
compared with scramble vector transfectant (p < 0.05). d The schematic diagram for the mechanisms underlying XIAP BIR promotion of EGFR protein
translation and anchorage-independent growth in bladder cancer cell
EGFR overexpression , a possible association of XIAP
with EGFR overexpression has never been explored in
previous studies. We demonstrate here that XIAP is a
strong positive regulator of EGFR expression in human
bladder cancers and that XIAP BIR domain plays an
important role in the mediation of EGFR protein expression
by promoting its protein translation. We find that BIR
domain of XIAP inhibits the Rac1 protein expression, which
leads to the decrease of PP2A phosphorylation at Tyr307,
in turn resulting in the activation of the PP2A activity and
consequently inactivating MAPKK/MAPK/c-Jun axis. The
inhibition of c-Jun further leads to a reduction of
miR200a transcription. Since miR-200a binds to the 3′-UTR
of EGFR mRNA and inhibits EGFR protein translation,
the attenuation of miR-200a expression by BIR domain of
XIAP results in the enhancement of EGFR protein
translation and increases the anchorage-independent growth of
the bladder cancer cells. This is the first demonstration
that XIAP promotes the anchorage-independent growth
of human bladder cancer cell via positive regulation of
There is increasing evidence showing that XIAP
functions much more than its ability to inhibit apoptosis. Our
recent studies have revealed several non-apoptosis-related
functions of XIAP via its RING domain, including the
upregulation of cyclin D1, while promoting bladder cancer
cell growth , and the promotion of F-actin formation
and colon cancer cell invasion via inhibition of
SUMOlation of RhoGDIα (Rho GDP-dissociation inhibitor α) at
lys-138 . Our most recent studies also reveal that the
RING domain of XIAP promotes Sp1-mediated
transcription of miR-4295, which targets the 3′ UTR of p63α
mRNA, for inhibiting p63α translation and enhancing
urothelial transformation . In the dissection of BIR
domain functions, we find that BIR domains can directly
bind to E2F1 (E2F transcription factor 1) and increase its
transactivation and cyclin E expression . In the current
studies, we identify a novel function of BIR domains in the
promotion of bladder cancer cell growth by upregulation
of EGFR expression. We find that knockdown XIAP
attenuated EGFR expression and the anchorage-independent
growth ability, while ectopic expression of XIAP BIR
domains in the XIAP knockdown cells restores the EGFR
level and the anchorage-independent growth ability.
The deficiency of EGFR expression and
anchorageindependent growth ability in the XIAP knockdown
cells is also reversed by ectopic expression of EGFR.
This is the first demonstration of XIAP BIR domains as
a potent positive regulator of EGFR expression, which in
turn promotes bladder cancer cell anchorage-independent
Nearly a quarter, between 20 and 30%, of human
bladder cancers are muscle-invasive bladder cancers, and half
of the patients diagnosed with muscle-invasive bladder
cancer will die from this aggressive disease within 5 years
[55, 56]. Thus, effective targeted therapeutic agents are
urgently needed. Due to their multidimensional roles in the
progression of cancers, XIAP and its family members have
emerged as attractive candidates for anti-cancer therapy
[57, 58]. A study shows that high level of XIAP expression
correlates with tumor differentiation and significantly
lower recurrence-free survival rates and independently
predicting the recurrence of non-muscular invasive
bladder cancer in a multivariate analysis . In addition to
XIAP, EGFR is also a well-known tumor therapeutic
target. EGFR is overexpressed in basal-like muscle-invasive
bladder cancers, which is sensitive to anti-EGFR therapy
. Other studies have shown that EGFR overexpressed
tumors are more sensitive to anti-IAPs therapy  and
XIAP also can lead to the resistance to anti-EGFR therapy
. Our studies here found that the overexpression of
EGFR was attributed to BIR domain of abnormally
expressed XIAP in bladder cancers (BCs), which offers an
exciting new opportunity for us to explore the potential
usage of XIAP BIR domain as a therapeutic target for
invasive BCs, which may avoid the issue of XIAP
related anti-EGFR therapy resistance, therefore in turn
helping to improve the clinical outcome of patients
with invasive BCs.
In this study, we also show a new mechanism for XIAP
BIR domain in regulating miR-200a through Rac1/PP2A/
MAPKK/MAPK/c-Jun axis. miR-200a, the anti-tumor
microRNA, is able to inhibit the epithelial-mesenchymal
transition and reverse the resistance to anti-EGFR therapy
[61, 62]. Our results showed that miR-200a could bind to
EGFR 3′-UTR and inhibit EGFR translation. Rac1, a
wellknown Rho GTPase, plays important roles in numerous
cellular functions . It had been demonstrated that
XIAP BIR domains are direct E3 ubiquitin ligases of Rac1
. Also, Rac1 has been reported to form a complex with
SET and decrease the PP2A activity through increased
PP2A phosphorylation level . We found here that
downregulation of XIAP resulted in an upregulated Rac1
and higher phosphorylation of PP2A, while ectopic
overexpression of Rac1 could also lead to a higher
phosphorylation level of PP2A. Further analysis revealed that the
knockdown of XIAP could lead to a high phosphorylation
level of PP2A, and subsequently activate the MAPK
kinase/MAPK pathway, leading to a high phosphorylation
level of c-Jun at ser63 and ser73, which could bind to the
miR-200a promoter region and promote the transcription
of miR-200a. Upon the treatment of cells with OA, a
PP2A specific inhibitor, the phosphorylation of MAPK
and MAPK kinases were markedly upregulated, while
EGFR expression was dramatically inhibited. Taken
together, we therefore demonstrate that BIR domains
regulated the miR-200a transcription through the
PP2A-related MAPK/c-Jun activation for the first time.
It was noted that an increased activation of MAPKs was
associated with a reduction of EGFR expression and
tumor growth. A similar observation has also been
reported in previous studies showing that the inhibited ERK
activity promotes the EGFR activation and augments
EGFR-driven motility of prostate cancer cells .
Although detailed mechanisms underlying this observation
are not explored yet, we anticipate that the comprehensive
feedback regulatory pathways might be involved. Further
elucidation of this issue will be helpful for understanding
the nature of XIAP in the regulation of BC growth.
In conclusion, we demonstrate a novel function of XIAP
BIR domain in regulating cancer cell
anchorageindependent growth ability through the upregulation of
EGFR translation via the inhibition of miR-200a
transcription through the Rac1/PP2A/MAPKK/MAPK/c-Jun axis.
This function provides new insight into the mechanisms
behind the XIAP regulating the cancer cell growth.
Altogether, the current studies provide very useful
information for new anti-XIAP therapeutic drug designs and
should in turn help to improve clinical outcomes of BC
patients with XIAP/EGFR overexpression.
BC: Bladder cancer; BIR: Baculovirus IAP repeat domains; CHX: Cycloheximide;
c-Jun(D): Dominant-negative c-Jun; EGFR: Epidermal growth factor receptor;
IAP: Inhibitors of apoptosis; MAPK: Mitogen-activated protein kinase;
MAPKK: MAPK kinase; NSCLC: Non-small-cell lung cancer; OA: Okadaic acid;
Pak1: p21-activated kinase-1; PP1: Protein phosphatase 1; PP2A: Protein
phosphatase 2; shRNA: Short hairpin RNA; TAF1β: Template activating Factor
1β; TGFα: Transforming growth factor α; UTR: Untranslated region; XIAP:
Xlinked inhibitor of apoptosis protein; ΔBIR: Deletion of N-terminal three BIR
domains; ΔRING: Deletion of C-terminal RING domain
This work was partially supported by the grants of NIH/NCI CA165980,
CA177665, and CA112557 and NIH/NIEHS ES000260, as well as Key Project of
Science and Technology Innovation Team of Zhejiang Province (2013TD10).
CSH, XRW, and HS designed the study. CH, XRZ, and GSJ detected the cells’
biological function, conducted the real-time PCR assays, carried out the Western
blot assays and Luciferase reporter assays, and performed the statistical analysis.
CH drafted the manuscript. XL, CL, JXL, HLJ, and JLZ helped to acquire the
experimental data. All authors read and approved the final manuscript.
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