XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell
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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.
Human XIAP was knockdown with short-hairpin RNA in two different bladder cancer cell lines, T24T and UMUC3. Two XIAP mutants, XIAP ∆BIR (deletion of N-terminal three BIR domains) and XIAP ∆RING (deletion of C-terminal RING domain and keeping the function of BIR domains), were generated to determine which domain is involved in regulating EGFR.
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.
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.
KeywordsXIAP BIR domain miRNA EGFR Rac1 Bladder cancer
Baculovirus IAP repeat domains
Epidermal growth factor receptor
Inhibitors of apoptosis
Mitogen-activated protein kinase
Non-small-cell lung cancer
Protein phosphatase 1
Protein phosphatase 2
Short hairpin RNA
Template activating Factor 1β
Transforming growth factor α
X-linked inhibitor of apoptosis protein
Deletion of N-terminal three BIR domains
Deletion of C-terminal RING domain
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, 7, 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, 12, 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 caspase-3 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, 24, 25, 26, 27, 28, 29]. Aberrant activation or expression of EGFR leads to the promotion of proliferation, cell motility, and invasive capacity of tumor [30, 31, 32, 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 anchorage-independent growth.
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, CA, USA).
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-∆RING 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
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 ∆BIR (deletion of N-terminal three BIR domains) and XIAP ∆RING (deletion of C-terminal RING domain and keeping the function of BIR domains) . The stable transfectants of ∆RING and ∆BIR in XIAP knockdown cells were established (Fig. 1a, b). Interestingly, while cells overexpressing XIAP ∆RING resumed EGFR protein levels in XIAP knockdown cells, the cells overexpressing ∆BIR 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 ∆RING 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 anchorage-independent growth in human bladder cancer cells.
XIAP BIR domain regulated the EGFR expression at translation level through increasing EGFR mRNA 3′ UTR activity
XIAP BIR domain-mediated inhibition of miR-200a was responsible for EGFR protein translational regulation by targeting EGFR mRNA 3′ UTR
XIAP BIR domain promoted miR-200a transcription by inhibiting c-Jun protein phosphorylation at Ser63/73
PP2A/MAPKK/MAPK axis was a BIR domain downstream effector mediated inhibition of c-Jun protein phosphorylation
To test whether inactivation of PP2A in UMUC3(shXIAP) cells mediates activation of MAPKKs/MAPKs/c-Jun, in turn reducing EGFR expression, we treated UMUC3(shXIAP/Vector) and UMUC3(shXIAP/∆RING) 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 expression.
XIAP BIR domain inhibited Rac1 expression and subsequently resulted in the downregulation of PP2A-C Phosphorylation at Tyr307
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 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 miR-200a 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 EGFR translation.
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 anchorage-independent 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 growth.
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 well-known 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 anchorage-independent 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.
We thank Dr. Benjamin Purow from University of Virginia Health System, Virginia, for a kind gift of EGFR 3′-UTR luciferase reporter.
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).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
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.
The authors declare that they have no competing interests.
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