The Interplay Between Eps8 and Irsp53 Contributes to Src-Mediated Transformation

Oncogene (2010) 29, 3977–3989 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation

P-S Liu1, T-H Jong2, M-C Maa3,4 and T-H Leu1,2,5

1Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 3Institute of Medical Science, China Medical University, Taichung, Taiwan; 4Institute of Molecular Systems Biomedicine, China Medical University, Taichung, Taiwan and 5Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan

As an oncoprotein, Eps8 participates in v-Src-induced Introduction cellular transformation. To delineate the underlying mechanism, we conducted a yeast two-hybrid screening The actin cytoskeleton is a dynamic structure that has a and identified IRSp53S, a protein critical in cell central role in a spectrum of cellular processes involving mobilization, as one of the Eps8-binding partners from a mobility and membrane dynamics. Among a plethora human brain cDNA library. The association was mediated of actin-binding proteins, insulin/insulin-like growth by the multiple proline-rich regions of Eps8 and the factor I receptor tyrosine kinase substrate IRSp53 C-terminal SH3-WWB containing domains of IRSp53S. (Yeh et al., 1996) turns to be linked to the Arp2/3- In this study, we observed that Eps8 modulated the mediated actin filament assembly as well as formation of expression of IRSp53 in v-Src-transformed cells (IV5), plasma membrane protrusions. IRSp53, also known raising the question of whether Eps8/IRSp53 interaction as brain-specific angiogenesis inhibitor 1-associated was crucial in carcinogenesis. To address this issue, we protein 2 (Abbott et al., 1999; Oda et al., 1999), is a generated IV5-expressing irsp53 siRNA cells. Attenuation founding member of a newly discovered family of of IRSp53 reduced cell proliferation of IV5 in culture dish proteins, including missing-in-metastasis (MIM) and and tumor formation in mice, which could be partly actin-bundling protein with brain-specific angiogenesis rescued by ectopically expressed human IRSp53S. In inhibitor 1-associated protein 2 homology (Scita et al., addition, IRSp53 knockdown impaired activity of phos- 2008). In addition to the originally identified 53-kDa phatidylinositol 3-kinase (as reflected by Pi-Ser473 AKT) protein IRSp53 (designated IRSp53S from now on), and Stat3 (as reflected by Pi-Tyr705 Stat3), and reduced there are at least three other human isoforms (desig- cyclin D1 expression that culminated to impede G1-phase nated IRSp58M, IRSp53T and IRSp53L) have been cell-cycle progression. Ectopically expressed human identified whereas only three murine homologues (that IRSp53S, but not its Eps8-binding defective mutants is, mIRSp53S, mIRSp58M and mIRSp53T) have been (that is, D363 and PPPDA), rescued these defects and detected (Miyahara et al., 2003). Interestingly, IRSp53S partly restored cell proliferation. Remarkably, through is suggested to be a candidate disease sensor of neuro- activation of Src, EGF increased the formation of Eps8/ degenerative polyglutamine (polyQ) expression (Okamura- IRSp53 complex and Stat3 activation in HeLa cells. With Oho et al., 1999). these results, we show for the first time that IRSp53, Members of IRSp53 contain identical N-terminal 511 through its interaction with Eps8, not only affects cell amino acids followed by unique sequences of 9–41 migration but also dictates cellular growth in cancer cells. amino-acid residues. In accordance to its role as an Oncogene (2010) 29, 3977–3989; doi:10.1038/onc.2010.144; adaptor protein, IRSp53 comprises an N-terminal Rac- published online 26 April 2010 binding domain (residues 1–228) that resides in IRSp53- MIM domain (residues 1–250), a central Cdc42/Rac Keywords: IRSp53; Eps8; v-Src; Stat3; oncogenic proteins; interactive binding motif followed by an SH3 domain, cancer cells WW-binding (WWB) domain and PDZ-binding motif at the C terminus. Interestingly, the amino-acid sequence within IRSp53 WWB (that is, PPPDY) is similar to the Eps8 SH3-binding consensus PXXDY (‘X’ indicates any amino-acid residue; Mongiovi et al., 1999). Proteins that Correspondence: Professor T-H Leu, Department of Pharmacology, interact with IRSp53 can be roughly divided into two College of Medicine, National Cheng Kung University, No. 1 groups. Although insulin receptor and brain-specific University Road, Tainan 70101, Taiwan. angiogenesis inhibitor (G protein-coupled receptor) E-mail: [email protected] or Professor M-C Maa, Institute belong to the first group of transmembrane receptors, of Medical Science, China Medical University, Taichung, Taiwan. E-mail: [email protected] the Rho family GTPases (that is, Rho, Rac and Cdc42) Received 9 November 2009; revised 7 March 2010; accepted 2 April and effectors (that is, mDia, WAVE2 and Mena) 2010; published online 26 April 2010 involved in stimulus-mediated actin and microtubule IRSp53 is critical in Src-mediated transformation P-S Liu et al 3978 cytoskeletal reorganization belong to the second one. Results Through its Rac-binding and SH3, IRSp53 interacts with Rac and WAVE2, respectively. Behaving Proline-rich sequences of Eps8 interact with the as a link between Rac and WAVE2, IRSp53 is C-terminal SH3-WWB domains of IRSp53 involved in lamellipodium formation. Interestingly, the Overexpression of Eps8, but not its N-terminal PH- crystal structure of IRSp53-MIM reveals an a-helical truncated mutant, induced cell transformation of coiled-coil domain (Millard et al., 2005; Lee et al., 2007) C3H10T1/2 cells (Maa et al., 2001). Using the N-terminal that is shown to possess actin filament-bundling PH-containing region of Eps8 (N-Eps8, residues 1–424) as (Millard et al., 2005), membrane-binding and mem- a bait, we isolated 211 Trp/Leu/His/LacZ all positives brane-deforming abilities (Suetsugu et al., 2006; Mattila from a human brain cDNA library screen of B2.5 Â 107 et al., 2007). clones. Purified DNA from anyone of the positives Eps8 is another IRSp53-binding protein. Its inter- complemented the bait N-Eps8 vector, but not the empty action with IRSp53 reinforces Rac activation and vector, to give Trp þ /Leu þ /His þ /LacZ þ . DNA sequen- cell migration in human fibrosarcoma cells (Funato cing revealed one of these N-terminal Eps8-binding et al., 2004). This result is consistent with the identifi- proteins was the human isoform of the already identified cation of Eps8 as an actin capper, which is capable hamster IRSp53S (Yeh et al., 1996). The schematic of regulating actin-based motility (Disanza et al., 2004). drawings of Eps8 and IRSp53S are shown in Figure 1a. Furthermore, formation of Eps8/SOS/Abi-1 complex To determine the region of Eps8 to associate with mediates Ras-induced Rac activation (Scita et al., 1999). IRSp53S, we fused a series of Eps8 deletion mutants to Unlike the involvement of Eps8 in cytoskeletal reorga- the GAL4 DNA-binding domain in pGBKT7 vector, nization is gradually disclosed, the implication of whereas we fused the full-length IRSp53S to GAL4 Eps8 in tumorigenesis is well established. Elevated activation domain in pGADT7 vector. Yeasts that harbor expression of Eps8 was observed in v-Src transformed pGBKT7-Eps8-proline-rich region (that is, Eps8-D424, (IV5) cells (Gallo et al., 1997; Maa et al., 1999), and Eps8-D561 and Eps8-CT) and pGADT7-IRSp53S can in a variety of human tumors including colorectal give Trp þ /Leu þ /His þ /LacZ þ (Figure 1b). Vice versa, (Maa et al., 2007), cervical (Chen et al., 2008), pituitary yeasts that harbor pGBKT7-Eps8- and pGADT7- (Xu et al., 2009) and oral cancers (Yap et al., 2009). IRSp53S-derived clones encoding both C-terminal SH3 Overexpression of Eps8 conferred the ability of fibro- and WWB domains (that is, IRSp53S-86-521, IRSp53S- blasts to form focus in culture dish and grow tumor 246-521, IRSp53S-305-521 and IRSp53S-376-521) can in mice (Maa et al., 2001), corroborating the onco- give Trp þ /Leu þ /His þ /LacZ þ (Figure 1c). Notably, genic potential of Eps8. In agreement, Eps8 attenua neither SH3 domain alone nor the C-terminal WWB tion retarded cellular growth in IV5 cells (Leu containing sequences is sufficient for Eps8 binding et al., 2004) and human cancer cells (Maa et al., 2007; (Figure 1c). As Eps8 participates in v-Src-mediated cell Chen et al., 2008; Xu et al., 2009). Remarkably, Eps8 is proliferation (Leu et al., 2004), we wonder whether Eps8/ also a prognostic factor for cervical cancer. This can be IRSp53 interaction has any role in v-Src-mediated evidenced by the observation that early-stage cervical mitogenesis. To address this issue, we first need to confirm cancer patients with higher Eps8 expression tend to have the interaction between Eps8 and IRSp53 in v-Src- parametrial invasion, lymph node metastasis and transformed cells (IV5). As shown in Figures 1d and e, decreased survival rate (Chen et al., 2008). the 53 and 58 kDa proteins of IRSp53 were observed in Despite IRSp53 serves as a physical linker between IV5 cells. Reverse transcription (RT)–PCR analysis of membrane and actin dynamics, its role in cell proli- IV5 mRNA revealed the presence of both mIRSp53S and feration and oncogenesis is still unclear. In this study, mIRSp53T (data not shown). Interestingly, the 97-kDa we observed that eps8 siRNA not only suppressed isoform of Eps8 was the major protein detected in the Eps8 expression, but also decreased the level of IRSp53 IRSp53 immunoprecipitates of IV5 cells (Figure 1d). in v-Src transformed IV5 cells. With yeast two-hybrid When Eps8 was immunoprecipitated from IV5 trans- assays, the proline-rich sequences and the C-terminal fected with various plasmid DNA encoding Myc-tagged SH3-WWB-containing region were mapped as the human IRSp53S (IRSp53S), C-terminal truncated binding sites in Eps8 and IRSp53S, respectively. IRSp53S (D363) or Myc-IRSp58M (IRSp58M), in Attenuation of IRSp53 suppressed cell proliferation of addition to endogenous IRSp53, both Myc-IRSp53S IV5 in both culture dish and soft agar and tumor and Myc-IRSp58M, but not Myc-D363, were detected formation in mice, which could be rescued by ectopically in Eps8 immunocomplexes (Figure 1e). These results expressed human IRSp53S but not the one with further support the importance of C-terminal SH3-WWB C-terminal deletion or mutation in the WWB domain. domains of IRSp53 in Eps8 binding. Finally, we presented evidence indicated that Src- mediated Eps8/IRSp53 interaction resulted in activation of AKT (that is, Pi-Ser473 AKT), ERK and Stat3 Eps8 modulates the expression of IRSp53 required (that is, Pi-Tyr705 Stat3) as well as enhancement of for v-Src-mediated transformation cyclin D1 in v-Src transformed cells and might Previously, we showed Eps8 was overexpressed in participate in EGF-mediated Stat3 activation in HeLa C3H10T1/2 cells expressing v-Src (IV5) and participated cells. And this is the first time that the mitogenic role of in Src-mediated transformation (Leu et al., 2004). To IRSp53 is revealed. compare the role of Eps8 and IRSp53 in mitogenesis, we

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3979

Figure 1 The C-terminal SH3-WWB domain of IRSp53 is responsible for its interaction with Eps8. (a) Schematic drawings of Eps8 (left) and IRSp53S (right). The relative position of irsp53 siRNA-targeting sequence is exhibited in the bottom. ‘Pro’ indicates the position of proline-rich regions in Eps8. The interaction of wild-type and deletion mutants of Eps8 with IRSp53S (b) or that of wild- type and deletion mutants of IRSp53S with Eps8 (c) was analyzed by yeast two-hybrid assay as described under Materials and methods section. The interaction between SV40 large T-antigen and p53 was a positive control (Control ( þ ). (d) IRSp53 was immunoprecipitated from IV5 cells (1 mg) with murine monoclonal anti-IRSp53 (IRSp53) or nonspecific (IgG) antibody crosslinked to protein A-sepharose. The immunoprecipitates and one-twentieth (that is, 50 mg) of IV5 lysate (input) were resolved by SDS–PAGE and western immunoblotted with antibodies as indicated. (e) IV5 was transiently transfected with a DNA plasmid encoding Myc- tagged human IRSp53S (IRSp53S), D363 (D363) or IRSp58M (IRSp58M). Equal amount (1 mg) of lysates prepared from these cells was immunoprecipitated with rabbit anti-Eps8 antibody crosslinked to protein A-sepharose followed by western immunoblotting with antibodies as indicated (left). One-twentieth (50 mg) of these lysates (input) was also western immunoblotted with anti-IRSp53 antibody (right). The star indicates the position of ectopically expressed Myc-IRSp53S, Myc-D363 or Myc-IRSp58M. It should be noted that a nonspecific protein (marked by an arrow) was also detected in each lane. introduced plasmids expressing nonspecific siRNA, eps8 IV5-based nonspecific siRNA transfected cells (Ctrl-1, siRNA, irsp53 siRNA (Figure 1a), irsp53 siRNA-b and Ctrl-2), IRSp53 was reduced in IV5-derived eps8 siRNA irsp53 siRNA-c (Supplemental Figure S1) into IV5 cells expressing cells (eps8 siRNA-1, eps8 siRNA-2) to obtain the corresponding siRNA cells. Western (Figure 2a). In contrast, the level of Eps8 was not analysis revealed the presence of IRSp53 in vector- affected in IV5-based irsp53 siRNA expressing cells transfected C3H10T1/2 cells (Neo) whose levels were (irsp53 siRNA-1, irsp53 siRNA-2) (Figure 2b). Given augmented in IV5 (Figure 2a). Compared to IV5 and that Eps8 was an oncoprotein, thereby we attempted

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3980 at least 50% less of tumors as compared to those inoculated with parental IV5 and Ctrl-1 cells (Table 1). In addition, IRSp53 attenuation reduced the growth rate of tumors (Figure 5). Furthermore, the eliminated oncogenesis of v-Src by irsp53 siRNA could be partly reversed by ectopically expressed human IRSp53S, but not by D363 (Table 1 and Figure 5).

IRSp53 is involved in the activation of AKT, Stat3 and the enhancement of cyclin D1 expression that accelerates cell-cycle progression in v-Src-transformed cells As an oncoprotein, v-Src facilitates G1-phase cell-cycle progression. Due to attenuation of Eps8 (Leu et al., 2004) or IRSp53 reduced v-Src-initiated mitogenesis, thus the role of Eps8 and IRSp53 in cell-cycle progression dictated by v-Src was attempted. As exhibited in Supplemental Figure S2, by flow cytometric analysis, knockdown of either IRSp53 or Eps8 slowed down G1-phase progression and reduced the population of cycling cells in S and G2/M phases. For 24-h-cultured IV5, attenuation of IRSp53 and Eps8 increased the B B Figure 2 Eps8 attenuation results in downregulation of IRSp53. proportion of G1 cells from 40 to 55% and 59%, (a) Lysates prepared from Neo, IV5 and IV5-derived cells respectively. Similar phenomenon was also observed in expressing nonspecific siRNA (Ctrl-1, Ctrl-2) or eps8 siRNA 48-h-cultured IV5 and its derived cells (Supplemental (eps8 siRNA-1,eps8 siRNA-2) were resolved by SDS–PAGE and Figure S2). On the basis of these findings, we concluded immunoblotted with antibodies as indicated. (b) Similar experiments were conducted in Neo, IV5 and IV5-derived cells expressing that both Eps8 and IRSp53 participated in v-Src- nonspecific siRNA (Ctrl-1, Ctrl-2) or irsp53 siRNA (irsp53 siRNA- accelerated cell-cycle progression. 1,irsp53 siRNA-2). Previously, we have shown that through AKT/ mTOR/Stat3 axis, Eps8 elevated the expression of cyclin D1 and FAK and facilitated cell-cycle progression in colon cancer cells (Maa et al., 2007). Given that the the involvement of its binding partner, IRSp53, in interaction between Eps8 and IRSp53 was pivotal in v-Src-mediated transformation. When the growth rate v-Src-initiated mitogenesis, one might wonder IRSp53 of Neo, IV5 and IV5-based Ctrl and irsp53 siRNA affected the same pathway regulated by Eps8. To answer cells was compared, attenuation of IRSp53 not only this question, we analyzed lysates harvested from Neo, reduced v-Src-mediated cell proliferation in culture dish IV5 and various aforementioned IV5-derived cells. As (Figure 3a), but also impaired v-Src evoked anchorage- shown in Figure 6, although the activity of Src (as independent growth in soft agar (Figure 3b). Similar reflected by Pi-Tyr416 Src) was not affected by irsp53 results were obtained in irsp53 siRNA-b-1, irsp53 siRNA, suppressed Pi-Ser473 AKT, Pi-Tyr705 Stat3 siRNA-b-2 and irsp53 siRNA-c-1, irsp53 siRNA-c-2 and reduced expression of cyclin D1 and FAK were cells (Supplemental Figure S1C and D). These findings observed. And ectopic IRSp53S, but not the Eps8- highlighted the importance of IRSp53 in transformation binding defective D363, restored these events. The elicited by v-Src. decrease of Pi-Ser473 AKT, Pi-Tyr705 Stat3, cyclin D1 and FAK was also observed in IV5-based irsp53 siRNA- Association between IRSp53 and Eps8 is critical for bandirsp53 siRNA-c cells (Supplemental Figure S1). v-Src-mediated transformation These findings indicated that the functional interaction Eps8/IRSp53 interaction reinforces Rac activation and between IRSp53 and Eps8 might be critical for Src to promotes cancer cell migration (Funato et al., 2004). trigger activation of both AKT and Stat3 required for Although attenuation of either protein greatly reduced the expression of cyclin D1 and FAK in v-Src- Src-mediated mitogenesis, one might wonder the role of transformed cells. Eps8/IRSp53 complex in this process. To answer this point, we generated irsp53 siRNA-1 cells with ectopic Ectopic IRSp53S restores cell proliferation and human IRSp53S, or its mutant lacking Eps8-binding anchorage-independent growth in Eps8-attenuated domain (D363), and performed a rescue experiment. As v-Src-transformed cells shown in Figure 4, ectopic IRSp53S, but not D363, In previous experiments, the importance of IRSp53 in increased anchorage-dependent and anchorage-indepen- Src-mediated transformation was revealed. Due to Eps8 dent growth of irsp53 siRNA-1 cells. Similar results were participated in Src-induced neoplasm (Leu et al., 2004) observed in irsp53 siRNA-2 cells (data not shown). and its attenuation caused IRSp53 downregulation Notably, mice inoculated with IRSp53 knockdown cells (Figure 2); thereby, we wondered whether the sup- (that is, irsp53 siRNA-1, irsp53 siRNA-2) developed pressed proliferation detected in IV5-based eps8 siRNA

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3981

Figure 3 Attenuation of IRSp53S reduces v-Src-mediated proliferation and anchorage-independent growth. (a) Neo, IV5 and IV5- derived Ctrl-1, Ctrl-2, irsp53 siRNA-1 and irsp53 siRNA-2 were plated at a density of 2 Â 105 cells per 60 mm dish at the beginning and then were counted by hemocytometer after 1-, 2- and 3-day culture and plotted. (b) For colony formation in soft agar, we applied 3 Â 103 cells of interest in the top agar at the beginning. After 2-week incubation, the colonies of the indicated cells were photographed ( Â 40), and the representatives were shown (up). The number of colonies (48 cells) was counted and plotted (bottom). **Po0.01 as compared with IV5. cells was attributable to IRSp53 reduction. To address higher than that of IRSp53S in irsp53 siRNA-1 cells, this issue, we individually introduced IRSp53S and its it was hardly detected in the Eps8 immunoprecipitates Eps8-binding defect mutant, that is D363, into IV5- (Figure 9a). Concurrently, PPPDA failed to restore based eps8 siRNA cells. Remarkably, ectopic IRSp53S, cell proliferation of IRSp53-attenuated (Figure 9b) but not D363, partly rescued the suppressed prolifera- and Eps8-attenuated (Figure 10) IV5 cells in both tion and anchorage-independent growth in eps8 siRNA culture dish and soft agar. Meanwhile, reduced Pi- cells (Figure 7). Further, western analysis disclosed that Ser473 AKT, Pi-Tyr705 Stat3 and cyclin D1 expression the reduced Pi-Ser473 AKT, Pi-Tyr705 Stat3, Pi-ERK in IRSp53-attenuated (Figure 9b) or Eps8-attenuated and the suppressed expression of cyclin D1 and FAK (Figure 10) IV5 cells was not effectively restored by caused by eps8 siRNA was also restored by ectopic ectopic PPPDA. These results further supported the IRSp53S, but not by D363 (Figure 8). These ﬁndings importance of Eps8/IRSp53 interaction in v-Src- indicated that Eps8 retained the ability to modulate the mediated mitogenesis. level of IRSp53 whose complex with Eps8 could trigger Src-mediated activation of AKT and Stat3, leading to the enhancement of cyclin D1 required for v-Src-evoked Src kinase activity participates in EGF-stimulated proliferation and tumorigenesis. Eps8/IRSp53 complex formation and Stat3 activation in HeLa cells As Src mediates Eps8/IRSp53 complex formation in IV5 Mutation of WWB domain abolishes the ability cells, we wonder whether its kinase activity is required of IRSp53S to restore cell proliferation and for this interaction. SU6656 is a potent inhibitor of Src anchorage-independent growth in IRSp53- family kinases (Blake et al., 2000; Bowman et al., 2001). or Eps8-attenuated v-Src transformed cells Its incubation with IV5 for 16 h reduced the expression The PPPDY sequence within the WWB domain of of Eps8 and the activation of AKT, ERK and Stat3 that IRSp53 is a potential binding site for Eps8-SH3 in vitro culminated in the decrement of cyclin D1 (Figure 11a). (Mongiovi et al., 1999). As D363 mutant lacks the entire By contrast, LY294002 and PD98059 that inhibited C-terminal SH3-WWB domains and other potential AKT and ERK, respectively, suppressed the level functional domains of IRSp53, we wondered the of Pi-Ser727 Stat3, but not of Pi-Tyr705 Stat3, and importance of WWB domain in IRSp53-participated only partially reduced the expression of cyclin D1 mitogenesis in IV5 cells. To address this point, we (Figure 11a). Remarkably, SU6656 also reduced the generated the IRSp53S mutant with PPPDA in its association between IRSp53 and Eps8 in IV5 cells WWB domain (designated as PPPDA) and reintroduced (Figure 11b). Thus, v-Src not only regulated the it into irsp53 siRNA-1 (Figure 9) and eps8 siRNA-1 expression of Eps8, but also its enzymatic activity could (Figure 10) cells. Though the level of PPPDA is much modulate the interaction between Eps8 and IRSp53.

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3982

Figure 4 Functional interaction between Eps8 and IRSp53 is required for v-Src-mediated cell proliferation in culture dish and colony formation in soft agar. (a) Lysates prepared from Neo, IV5 and IV5-derived Ctrl-1, Ctrl-2, irsp53 siRNA-1, irsp53 siRNA-2 and irsp53 siRNA-1 bearing either Myc-IRSp53S (irsp53 siRNA-1/IRSp53S-1, irsp53 siRNA-1/IRSp53S-2) or Myc-D363 (irsp53 siRNA-1/ D363-1, irsp53 siRNA-1/D363-2) were resolved by SDS–PAGE and immunoblotted with antibodies as indicated. Assays for proliferation of these cells in culture dishes (b) or soft agar (c) were performed as described in Figure 3. *Po0.05; **Po0.01 as compared with IV5. #Po0.05.

Table 1 Tumorigenicity of IRSp53-attenuated IV5 cells in Figure 12a, EGF elicited Src activation (as reﬂected by Pi-Tyr416 Src and Pi-Tyr861 FAK) and Stat3 Cell line Tumor frequencya phosphorylation on Tyr705 in HeLa cells. And con- v-Src transformed cells (IV5) 8/8 comitantly, the formation of Eps8/IRSp53 complex was Ctrl-1 8/8 also increased (Figure 12b). The deterioration of these irsp53 siRNA-1 4/8 events by SU6656 (Figures 12a and b) suggested that irsp53 siRNA-2 0/8 irsp53 siRNA-1/IRSp53S 8/8 Eps8/IRSp53-medicated Stat3 activation might also irsp53 siRNA-1/D363 4/8 occur in human cancer cells in response to EGF.

aApproximately 107 cells of interest in 0.1 ml of sterile PBS were injected subcutaneously into the hip of 4- to 6-week-old C3H/HeN mice. Nineteen days later, tumor frequency was expressed as number of mice with tumors/total number of mice. Discussion Attenuation of Eps8 decreased v-Src-induced mito- Given that all experiments concerning Eps8/IRSp53 genesis (Leu et al., 2004). In an attempt to delineate were conducted in ﬁbroblasts expressing v-Src, thereby the underlying mechanism mediated by Eps8, search for we would like to know whether Eps8/IRSp53 axis its interacting partners through a yeast two-hybrid was also present and acted as a Src-mediated down- screening from a brain cDNA library was prompted. stream signaling in human cancer cells. As shown In this study, we reported that IRSp53S was identiﬁed as

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3983

Figure 6 Eps8/IRSp53 functional interplay is required for increased Pi-Ser473 AKT, Pi-Tyr705 Stat3 and elevated expression of cyclin D1 in v-Src-transformed cells. Equal amount of lysates prepared from Neo, IV5 and IV5 derived Ctrl-1, Ctrl-2, irsp53 siRNA-1, irsp53 siRNA-2, irsp53 siRNA-1/IRSp53S-1, irsp53 siRNA-1/IRSp53S-2, irsp53 siRNA-1/D363-1 and irsp53 siRNA- 1/D363-2 was resolved by SDS–PAGE and immunoblotted with antibodies as indicated.

suggested that IRSp53 might be involved in Eps8- mediated mitogenesis (Figure 2). Indeed, attenuation of IRSp53 in IV5 cells hampered cell proliferation in culture dish or soft agar (Figures 3 and 4, and Supple- mental Figure S1), and tumor formation in mice (Table 1 and Figure 5). Reintroduction of human IRSp53S, but not its Eps8-binding defective mutant (that is, D363), partially rescued these defects (Table 1, Figures 4 and 5). Consistent with these biological responses, attenuation of either IRSp53 (Figure 6 and Supplemental Figure S1) or Eps8 (Figure 8) suppressed Pi-Ser473 AKT, Pi- Tyr705 Stat3 and the expression of cyclin D1 and FAK, leading to retarded G1-phase progression (Supplemental Figure S2). And these defects could be alleviated by ectopic IRSp53S, but not by D363 (Figures 6 and 8). Notably, overexpression of IRSp53S, but not of D363, Figure 5 Eps8/IRSp53 functional interplay is required for v-Src- rescued anchorage-dependent and anchorage-indepen- mediated tumor formation in syngenic mice. (a) Equal number of dent growth of Eps8-attenuated cells (Figure 7). Thus, cells (1 Â 107) from IV5 and IV5-derived Ctrl-1, irsp53 siRNA-1, an unperceived role of IRSp53 in v-Src/Eps8-mediated irsp53 siRNA-2, irsp53 siRNA-1/IRSp53S-1, irsp53 siRNA-1/ mitogenesis was for the ﬁrst time disclosed. D363-1 was injected subcutaneously into the hip of C3H/HeN There are two Eps8 isoforms, p97Eps8 and p68Eps8,in mice (n ¼ 8). One week later, the size of each tumor was measured Eps8 every 3 or 4 days and plotted as described under Materials and IV5 cells. Though p68 shares most of its C-terminal methods section. (b) Around 19 (IV5 and Ctrl-1) or 28 days (irsp53 sequences with that of p97Eps8 (Maa et al., 1999), its siRNA-1, irsp53 siRNA/IRSp53S-1 and irsp53 siRNA/D363-1), the function is still obscure. Interestingly, despite the tumors were excised, photographed (top), weighed and plotted C-terminal proline-rich sequences of Eps8 were able to (bottom). It should be noted that only 50% of the mice inoculated with irsp53 siRNA-1 or irsp53 siRNA-1/D363-1 developed tumor associate with IRSp53 in our yeast two-hybrid experi- Eps8 and none of the mice inoculated with irsp53 siRNA-2 did. The ments (Figure 1b and data not shown), p97 turned to weights of the tumors excised are mean±s.d. in two independent be the major proteins associated with IRSp53 in IV5 experiments. ***Po0.001. (Figure 1d). Consistently, the N-terminal proline-rich sequence spanning from aa 207 to aa 221, which was absent in p68Eps8 (Maa et al., 1999 and data not shown), an Eps8-binding protein, which associated with the was responsible for Eps8 interacting with IRSp53 in proline-rich sequences of Eps8 (Figure 1). And the promoting Rac activation and cell motility (Funato C-terminal SH3/WWB-containing sequence (aa 376–521) et al., 2004). And this Eps8/IRSp53 complex occurred of IRSp53S was found to be essential and sufﬁcient to at the leading edge of motile cells (Funato et al., 2004). interact with Eps8 (Figure 1). The Eps8-dependent In agreement, 261-p97Eps8 that lacked an intact PH enhancement of IRSp53 in v-Src transformed cells domain as well as the N-terminal proline-rich sequences

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3984

Figure 8 Ectopic IRSp53S, but not D363, compensates the signaling defects caused by Eps8 attenuation in v-Src-transformed cells. Equal amount of lysates prepared from Neo, IV5 and IV5- derived Ctrl-1, Ctrl-2, eps8 siRNA-1, eps8 siRNA-2 and eps8 siRNA-1 bearing either Myc-IRSp53S (eps8 siRNA-1/IRSp53S-1, eps8 siRNA-1/IRSp53S-2) or Myc-D363 (eps8 siRNA-1/D363-1, eps8 siRNA-1/D363-2) was resolved by SDS–PAGE and immunoblotted with antibodies as indicated.

implicated for Eps8 interaction. However, according to our results, the SH3 domain alone was insufﬁcient for IRSp53/Eps8 interaction in yeast (Figure 1c). Inter- estingly, a WWB sequence, that is, PPPDY, located at the C terminus of IRSp53, was a canonical Eps8 SH3- binding motif (that is, PXXDY; Mongiovi et al., 1999) and might contribute to Eps8 interaction. Indeed, IRSp53S containing the C-terminal SH3-WWB sequences, that is, aa 376–521, was capable to interact with Eps8 in yeast (Figure 1c). In agreement, IRSp53S lacked these two domains (that is, D363) failed to associate with Eps8 (Figure 1e) and rescue cell proliferation of Eps8- attenuated IV5 cells (Figure 7). Recently, the solution structure of Eps8 family SH3 (that is, Eps8L1 SH3) in Figure 7 Ectopic IRSp53S, but not D363, increases both complex with a peptide containing the PXXDY motif anchorage-dependent and anchorage-independent cell proliferation in Eps8-attenuated v-Src transformed cells. Equal amount of was resolved by NMR spectroscopy (Aitio et al., 2008). lysates prepared from Neo, IV5 and IV5-derived Ctrl-1, Ctrl-2, The formation of a hydrogen bond between the carboxyl eps8 siRNA-1, eps8 siRNA-2 and eps8 siRNA-1 bearing either group of E496 shared by Eps8 family SH3 and the Myc-IRSp53S (eps8 siRNA-1/IRSp53S-1, eps8 siRNA-1/IRSp53S- hydroxyl group of the aromatic ring of Y explained the 2) or Myc-D363 (eps8 siRNA-1/D363-1, eps8 siRNA-1/D363-2) was resolved by SDS–PAGE and immunoblotted with antibodies as indispensability of the tyrosine residue in PXXDY motif indicated (top). The star indicates the position of ectopically in Eps8 association. The inability of ectopic PPPDA expressed Myc-IRSp53S or Myc-D363. With the same methodo- to restore the growth defect of IRSp53-attenuated logy described in Figure 3, proliferation (middle) and colony (Figure 9) and Eps8-attenuated IV5 (Figure 10) further formation (bottom) of these cells were determined. **Po0.01 as supported the importance of Eps8/IRSp53 complex in compared with IV5. #P 0.05. o Src-mediated mitogenesis. Despite attenuation of either IRSp53 or Eps8 resulted failed to promote anchorage-independent growth and in suppressed proliferation and tumorigenesis in v-Src- tumor formation in mice (Maa et al., 2001). These transformed cells, there were substantial differences results highlighted the importance of membrane-bound between their exerted effects. For instance, mice Eps8 to interact with IRSp53 required for abnormal inoculated with Eps8-attenuated IV5 cells still grew cell proliferation. Remarkably, the association between tumors (data not shown), but reduced tumor incidence Eps8 and IRSp53 required the activation of Src (Figures was observed in mice inoculated with IRSp53-attenu- 11b, 12b). To date, only the SH3 domain of IRSp53 was ated IV5 cells (Table 1). To date, three IRSp53 isoforms

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3985

Figure 9 Ectopic PPPDA mutant neither associates with Eps8 nor increases both anchorage-dependent and anchorage-independent cell proliferation in IRSp53-attenuated v-Src transformed cells. The generation of irsp53 siRNA-1/IRSp53S-1 and irsp53 siRNA-1 bearing Myc-PPPDA (irsp53 siRNA-1/PPPDA-1, siRNA-1/PPPDA-2) were described above and/or under Materials and methods section. (a) Lysates prepared from irsp53 siRNA-1/IRSp53S-1 (IRSp53S) and irsp53 siRNA-1/PPPDA-1 (PPPDA) were western immunoblotted with antibodies as indicated (top) or immunoprecipitated with Eps8 antibody followed by western immunoblotted with Myc antibody or Eps8 antibody (bottom). The star indicates the position of ectopically expressed Myc-IRSp53S or Myc-PPPDA. (b) Equal amount of lysates prepared from Neo, IV5 and IV5-derived Ctrl-1, irsp53 siRNA-1, irsp53 siRNA-1/PPPDA-1 and irsp53 siRNA-1/PPPDA-2 was resolved by SDS–PAGE and immunoblotted with antibodies as indicated (top). The star indicates the position of ectopically expressed Myc-PPPDA. With the same methodology described in Figure 3, proliferation (middle) and colony formation (bottom) of these cells were determined. ***Po0.001 as compared with IV5.

(that is, mIRSp53S, mIRSp53T and mIRSp58M) with IRSp53L, insulin-like growth factor I augmented the different C-terminal amino-acid residues (Miyahara phosphorylation of IRSp53T (Okamura-Oho et al., et al., 2003) were reported in murine cells. As insulin 2001). Thus, it was likely that each IRSp53 isoform increased the phosphorylation of IRSp53S and had its own regulation and physiological implication.

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3986

Figure 11 Eps8/IRSp53 functional interplay is sensitive to SU6656 in v-Src-transformed cells. (a) Equal amount of lysates prepared from Neo and IV5 treated with dimethylsulfoxide (DMSO) (Veh), LY294002 (LY, 10 mM), PD98059 (PD, 10 mM)or SU6656 (SU, 5 mM) for 16 h was resolved by SDS–PAGE and immunoblotted with antibodies as indicated. (b) Equal amount (1 mg) of lysates prepared from IV5 treated with DMSO (Veh) or SU6656 (SU) as described in panel a were immunoprecipitated with anti-Eps8 antibody. The Eps8 immunocomplexes were resolved by SDS–PAGE and immunoblotted with anti-IRSp53 (left, top) and anti-Eps8 (left, bottom) antibodies. One-twentieth (50 mg) of these lysates (input) was also western immunoblotted with anti-IRSp53 Figure 10 Ectopic PPPDA mutant could not restore both antibody (right). anchorage-dependent and anchorage-independent cell proliferation in Eps8-attenuated v-Src transformed cells. Equal amount of lysates prepared from Neo, IV5 and IV5-derived Ctrl-1, eps8 Despite IRSp53S was the only one identiﬁed as the siRNA-1 and eps8 siRNA-1 bearing Myc-PPPDA (eps8 siRNA-1/ Eps8-interacting protein in our yeast two-hybrid screen, PPPDA-1, eps8 siRNA-1/PPPDA-2) was resolved by SDS–PAGE and immunoblotted with antibodies as indicated (top). With the we did not exclude the possibility that irsp53 siRNA same methodology described in Figure 3, proliferation (middle) might also suppress the expression of the other two and colony formation (bottom) of these cells were determined. IRSp53 isoforms and deteriorate their mediated signal- ***Po0.001 as compared with IV5. ing required for v-Src-triggered oncogenesis.

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3987 dependent and might be a physiological event relaying EGF signaling. At present, research regarding the function of IRSp53 was all focused on its modulation of actin dynamics and cell mobility. Here, we presented evidence indicating the involvement of IRSp53 in v-Src-elicited proliferation and tumorigenesis, which required its interplay with Eps8. In addition, this Src-mediated Eps8/IRSp53 association also occurred in EGF-stimulated HeLa cells. This prompted IRSp53 to be a potential oncoprotein and a therapeutic target for cancer treatment.

Materials and methods

Cells and lysate preparation Murine C3H10T1/2 fibroblast and human cervical carcinoma HeLa cells were used in this study. C3H10T1/2 cells transfected with plasmid DNA encoding v-Src (IV5) or vector alone (Neo) and IV5-derived eps8 siRNA-expressing cells were described previously (Leu et al., 2004). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cultured cells were lysed in modified RIPA buffer as described previously (Maa and Leu, 1998) and protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA).

Antibodies, immunoprecipitation and immunoblotting Antibodies against Eps8 (C-Eps8) (Maa et al., 1999) or FAK (CTF3) (Maa and Leu, 1998) were described previously. The primary antibodies used were actin, Eps8, FAK (A-17), Myc (9E10), ERK1 and Stat3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Eps8 and IRSp53 (BD Transduction Figure 12 EGF-increased formation of Eps8/IRSp53 complex and Laboratories, Franklin Lakes, NJ, USA); Pi-Tyr416 Src and Pi-Tyr705 Stat3 are sensitive to SU6656 in HeLa cells. Serum-starved Pi-Tyr861 FAK (BioSource International, Camarillo, CA, HeLa was pretreated with dimethylsulfoxide (DMSO) (Veh) or USA); Pi-Tyr705 Stat3, Pi-Ser727 Stat3, AKT, Pi-Tyr473 SU6656 (SU, 5 mM) for 20 min, and then cells were stimulated with AKT and cyclin D1 (Cell Signaling, Danvers, MA, USA). EGF (100 ng/ml) for 15 min. (a) Equal amount of lysates was analyzed Immunoprecipitation of Eps8 was carried out by using rabbit directly by SDS–PAGE and immunoblotted with antibodies as anti-Eps8 antibody (Santa Cruz Biotechnology) crosslinked to indicated. It should be noted that a nonspecific protein (marked by protein A sepharose (using dimethyl pimelimidate-coupling an arrow) was also detected by IRSp53 antibody in this particular method (Harlow and Lane, 1988)). For precipitation of experiment. The relative amounts of Pi-Tyr705 Stat3 and Stat3 were quantified by densitometer scanning, and the ratio was indicated on IRSp53, murine monoclonal anti-IRSp53 and control immuno- the bottom when EGF-untreated Veh was given a value of 1.0. (b)Ina globulin (IgG) were crosslinked to protein A sepharose pre- parallel experiment as described above, equal amount (1 mg) of lysates coupled to rabbit anti-mouse IgG (Jackson ImmunoResearch was immunoprecipitated with Eps8 antibody and immunoblotted with Laboratories, West Grove, PA, USA) as described above. anti-IRSp53 and anti-Eps8 antibodies. Western immunoblots were performed with respective antibody and detected by chemiluminescence as described previously Activation of Stat3 is required for v-Src-mediated (Maa and Leu, 1998). transformation (Martin, 2001; Silva, 2004). In addition to v-Src-transformed cells, Src-dependent Stat3 acti- Yeast two-hybrid assays vation has been implicated in the mitogenesis of c-DNA encoding N-terminal Eps8 fragment (aa 1–424) were EGF family receptor tyrosine kinases in human cancer cloned into pGBKT7 (a Gal4 DNA-binding domain-contain- cells (Silva, 2004). Detection of IRSp53 in Eps8 ing plasmid) and was introduced into Saccharomyces cerevisiae immunocomplexes from IV5 (Figure 11b) and HeLa strain AH109 according to the manual of the Matchmaker (Figure 12b) indicated that Eps8/IRSp53 complex was GAL4 Two-Hybrid System 3 (CLONTECH Laboratories, not only present in murine fibroblasts expressing Palo Alto, CA, USA). Pretransformed human brain Match- v-Src, but also existed in human cancer cells. Markedly, maker cDNA library (constructed in pACT2 plasmid, containing GAL4 activation domain, and was transformed into S. simultaneously increased Eps8/IRSp53 complex cerevisiae strain Y187) was purchased from Clontech Labora- (Figure 12b) and elevated Pi-Tyr705 Stat3 (Figure 12a) tories Inc. The mating protocol was performed as described in were observed in EGF-stimulated HeLa and was the provided manual. Near 25 million interactions were tested. sensitive to SU6656 treatment. These findings suggested The DNA fragments of the positive clones were amplified by the association between Eps8 and IRSp53 was Src- PCR using the primers provided in the kit. The resulting

Oncogene IRSp53 is critical in Src-mediated transformation P-S Liu et al 3988 sequences were used to identify the corresponding gene in the achieved by RT–PCR amplification using SW620 mRNA as GenBank Database (NCBI, Bethesda, MD, USA). a template and confirmed by DNA sequencing. For protein interaction in yeast, we mated yeast strain The generation of IV5 cells stably expressing eps8 siRNA AH109 harboring cDNA fragments encoding various parts of (eps8 siRNA) was described previously (Leu et al., 2004). To Eps8 in pGBKT7 with yeast strain Y187 containing full- generate cells stably expressing irsp53 siRNA (irsp53 siRNA) length IRSp53S cDNA in pGADT7 (pGADT7-IRSp53S). or the nonspecific siRNA (Ctrl), we transfected IV5 cells with Vice versa, yeast Y187 strain containing cDNA fragments DNA of pS-mIRSp53 or the negative control pSilencer encoding various portions of IRSp53S in pGADT7 was mated plasmid (which contains sequences not present in mouse with yeast strain AH109 containing full-length Eps8 cDNA genome and provided by Ambion Inc.) by Lipofectamine in pGBKT7. Plus method (Invitrogen, Carlsbad, CA, USA) followed by hygromycin selection (Leu et al., 2004). To generate irsp53 siRNA cells and eps8 siRNA cells expressing human IRSp53S, Tumorigenicity D363 mutant or PPPDA mutant, we co-transfected irsp53 Approximately 107 cells in 0.1 ml of sterile phosphate-buffered siRNA cells and eps8 siRNA cells with pBabe(puro) plus either saline were injected subcutaneously into the hip of 4- to pCMV-myc-IRSp53S, pCMV-myc-D363 or pCMV-myc- 6-week-old mice (that is, C3H/HeN). Mice were checked every IRSp53S(PPPDA) followed by puromycin (2 mg/ml) selection 3 or 4 days after 1-week inoculation and the tumor formed was (Maa et al., 2007). measured as described previously (Maa et al., 2001). After 19 or 28 days, we killed mice and excised and weighed the tumors. Statistic analysis The mouse experiments were performed according to the Each experiment was performed at least three times. Data are ethical guidelines for laboratory animal use and approved by presented as mean±s.d. from a representative triplicate the Institutional Animal Study Committee at National Cheng experiment. Unless indicated, the significance of difference Kung University. was assessed by Student’s t-test as compared to that of IV5. Bonferroni correction was used for controlling type I error in Plasmid construction and cell lines generation multiple comparisons. To construct a plasmid (pS-mIRSp53) expressing murine irsp53 siRNA, we synthesized, annealed and ligated two comple- mentary isrp53-containing oligonucleotides (sense strand, Conflict of interest 50-GATCCGGCTATTTCGATGCTCTGGTTCAAGAGACC 0 AGAGCATCGAAATAGCCTTTTTTGGAAA-3 ; and anti- The authors declare no conflict of interest. sense strand, 50-AGCTTTTCCAAAAAAGGCTATTTCGAT GCTCTGGTCTCTTGAACCAGAGCATCGAAATAGCCG-30) into pSilencer hygro vector (Ambion Inc., Austin, TX, USA) according to the manufacturer’s recommendation. The con- Acknowledgements struction of human IRSp53S-expressing plasmid (pCMV-myc- IRSp53S) was conducted by PCR amplification using This work was supported by National Science Council grants pGADT7-IRSp53S DNA as a template and confirmed by to M-CM (NSC98-2311-B-039-002-MY3) and T-HL (NSC97- DNA sequencing. To generate a plasmid (that is, pCMV-myc- 2320-B-006-024-MY3). Additional support came from NHRI IRSp53S (PPPDA)) expressing Y to A mutation in the WWB (NHRI-EX-98-9828BI to T-H Leu), Taiwan Department of of IRSp53S, we performed site-directed mutagenesis by PCR Health Clinical Trial and Research Center of Excellence reaction using pCMV-myc-IRSp53S DNA as a template and a (DOH99-TD-B-111-004 to M-C Maa) and China Medical pair of primers (sense strand, 50-GCCATCCCACCCCCCGA University (CMU98-C-05 to M-C Maa). The DNA constructs TGCCGGCGCCGCCTCCCGG-30; and antisense strand, of pLKO.1-mirsp53-b and pLKO.1-mirsp53-c were provided 50-CCGGGAGGCGGCGCCGGCATCGGGGGGTGGGAT by the National RNAi Core Facility located at the Institute of GGC-30). The pCMV-myc-D363 was constructed by XhoI Molecular Biology/Genomic Research Center, Academia digestion of pCMV-myc-IRSp53S DNA to delete the 30 Sinica, supported by the National Research Program for nucleotides encoding aa 364–521. The construction of human Genomic Medicine Grants of NSC (NSC 94-3112-B-001-003 IRSp53M-expressing plasmid (pCMV-myc-IRSp58M) was and NSC 94-3112-B-001-018-Y).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene