Oncogene (2008) 27, 2525–2531
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ORIGINAL ARTICLE
Protein tyrosine phosphatase PTPN13 negatively regulates Her2/ErbB2 malignant signaling J-H Zhu1, R Chen1, W Yi1, GT Cantin2, C Fearns1, Y Yang3, JR Yates III2 and J-D Lee1 1 Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA; 2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA and 3Johnson and Johnson Pharmaceutical Research and Development, San Diego, CA, USA
Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors. Oncogene (2008) 27, 2525–2531; doi:10.1038/sj.onc.1210922; published online 5 November 2007 Keywords: PTPN13; Her2; dephosphorylation; invasion and negative feedback
Introduction Her2/ErbB2 together with Her1/ErbB1, Her3/ErbB3 and Her4/ErbB4 are members of epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. Activation of Her2 triggers autophosphorylation of specific tyrosine residues within its cytoplasmic
domain, consequently activating many mitogenic/oncogenic intracellular signaling cascades such as PI(3)K/ AKT and MAP kinases, and inducing cellular processes such as cell migration, invasion, differentiation and proliferation (Hackel et al., 1999; Gensler et al., 2004). Overexpression of Her2 has been found in a variety of solid tumors such as bladder, breast and ovarian cancers (Menard et al., 2004), and numerous reports from in vitro and mouse models have demonstrated that continued activation of Her2 has a causal role in tumorigenesis and leads to tumor progression (Slamon et al., 1987; Moody et al., 2002; Menard et al., 2004). Phosphorylation of tyrosine residues is usually a critical step for regulating activities of certain cellular proteins and subsequent activation/deactivation of their downstream signaling events. This type of modification is generally controlled by the balance between the opposing activities of protein kinases and tyrosine phosphatases (PTPs). In recent years, much attention has been given to the regulation by PTPs and their physiological functions (Gil-Henn and Elson, 2003). PTPs are broadly divided into single transmembrane receptor-like forms and nonreceptor forms. Recent evidences have shown that PTPs can function as tumor suppressors but some PTPs such as SHP2 can be oncogenic (Ostman et al., 2006; Tonks, 2006). Activating mutations in SHP2 have been identified in various tumors, particularly in leukemias (Tartaglia et al., 2003; Tartaglia and Gelb, 2005; Ostman et al., 2006). Through screening a phosphatase siRNA library, we found protein tyrosine phosphatase PTPN13 (also known as FAP-1, PTP-BAS and PTPL1) was involved in the regulation of phosphorylation of the Her2 signaling domain. PTPN13, a nonreceptor PTP, is the largest of known PTPs (Alonso et al., 2004). PTPN13 has been previously suggested to have tumor-suppressive activity (Wang et al., 2004; Ostman et al., 2006; Yeh et al., 2006) and mutations in PTPN13 have been reported in colon and hepatocellular cancers (Wang et al., 2004; Yeh et al., 2006). In this study, we demonstrated a role of PTPN13 in regulating Her2 activity. Results
Correspondence: Professor J-D Lee, Department of Immunology, The Scripps Research Institute, IMM-12, 10550 N. Torrey Pines Road., La Jolla, CA 92037, USA. E-mail:
[email protected] Received 19 June 2007; revised 8 October 2007; accepted 11 October 2007; published online 5 November 2007
PTPN13 knockdown by siRNA increases the phosphorylation of Her2 signaling domain A phosphatase siRNA library was used to screen for phosphatase(s) that affected Her2 phosphorylation.
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Among 193 phosphatases (40 protein tyrosine phosphatases) screened by siRNA knockdown, knockdown of 7 protein tyrosine phosphatases (PTPs) significantly increased phospho-Her2 levels after growth factor stimulation (Table 1). We further confirmed these data by increasing expression of these PTPs by transfection of expression plasmids and found that only increased expression of PTPN13 and PTPN14 can reduce the Her2 phosphorylation induced by growth factor (No data is available about increased expression of PTPRD since expression plasmid of this gene is toxic to cells). We further analysed the phosphatase activity of both PTPN13 and PTPN14 and detected phosphatase activity only in PTPN13 but not in PTPN14 (Table 1). Thus, we focused our effort on PTPN13. PTPN13 knockdown by siRNA significantly increased phosphorylation of the Her2 signaling domain after growth factor stimulation (Figure 1a and Supplementary Figure S1B). As controls, mock transfection and siRNA-mediated big mitogen-activated kinase 1 (BMK1) knockdown had no effect on Her2 phosphorylation. The effect of PTPN13 knockdown on Her2 phosphorylation was validated by two individual siRNAs against PTPN13 (Supplementary Figure S2). Time course experiments showed that EGF-induced Her2 phosphorylation was enhanced by PTPN13 knockdown at all time points tested (Figure 1b), but the augmentation of Her2 phosphorylation was more significant at the earlier time points (5 and 15 min) indicating that PTPN13 has a considerable role in inhibiting the early (5 and 15 min) rather than late (60 min) growth factor-induced Her2 signaling. Accordingly, activation of downstream Akt and Stat3 was elevated by PTPN13 knockdown, particularly at the earlier time points (5 and 15 min). A slight increase in Erk1/2 activation at the earlier times was also observed in the PTPN13-deficient cells (Figure 1b). Increased expression of cellular PTPN13 inhibits Her2 phosphorylation, and Her2 forms a stable complex with a phosphatase inactive form of PTPN13 We constructed a catalytically inactive mutant of PTPN13 by mutating aspartic acid 2359 in the phosphatase domain to alanine. As predicted, PTPN13D2359A had little phosphatase activity compared with the wild-type PTPN13 (Figure 2a). We next tested whether expression of wildtype PTPN13 or inactive PTPN13D2359A had any effect on Her2 phosphorylation. Compared to the mock-transfected and empty vector-transfected controls, increased
expression of wild-type PTPN13 inhibited Her2 phosphorylation after EGF stimulation, while the mutant PTPN13D2359A promoted it (Figure 2b). This modulation of the phosphorylation of Her2 raised the question whether PTPN13 can form a complex with Her2. To address this question, an in vitro binding experiment was performed using either wild-type PTPN13 or mutant
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Figure 1 PTPN13 knockdown by siRNA increases the phosphorylation of Her2 signaling domain. (a) HeLa cells were transfected with siRNA against BMK1 (siBMK1) or against PTPN13 (siPTPN13). Mock-transfected or siBMK1-transfected HeLa cells were used as controls. Forty-eight hours post transfection, the cells were serum starved overnight followed by 10 ng ml 1 EGF stimulation for 15 min. The levels of phospho-Her2 (P-Her2), Her2, BMK1, PTPN13 and GADPH in these cells were analysed by western blot using specific antibodies (described in Materials and methods) as indicated. (b) HeLa cells were mock transfected or transfected with siPTPN13 and starved as in (a). These cells were stimulated with 10 ng ml 1 EGF for 0, 5, 15, 30 and 60 min as indicated and the levels of P-Her2, Her2, phospho-Akt, phosphoStat3, phospho-Erk1/2 and GADPH in these cells were examined by western blot.
Table 1 Effect of seven protein tyrosine phosphatases on Her2 phosphorylation
siRNA knockdown Overexpression Phosphatase activity
PTPN3
PTPN13
PTPN14
PTPRB
PTPRD
PTPRE
PTPRN2
m
m k +
m k ND
m
m NA NA
m
m
+
ND
+
+
Abbreviations: NA, data not available due to toxic plasmid; ND, activity not detectable. HeLa cells were stimulated 15 min with 10 ng ml 1 EGF. Phosphatase activity was assayed as described in Materials and methods. m, increased Her2 phosphorylation compared to control; k, decreased Her2 phosphorylation compared to control; , no change of Her2 phosphorylation compared to control; +, activity detectable. Oncogene
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PTPN13D2359A. Indeed, by immunoprecipitation, we showed that catalytically inactive PTPN13D2359A, formed a much more stable complex with Her2 protein than wildtype PTPN13 did (Figure 2c), suggesting that the inactive form of PTPN13 not only loses its phosphatase activity but also may protect the dephosphorylation of the Her2 signaling domain by stably interacting with Her2 thereby preventing the access of other cellular phosphatase(s) to the Her2 signaling domain. The growth factor-mediated phosphorylation of PTPN13 is involved in the dephosphorylation of Her2 by PTPN13 Through functional phosphoproteomic analysis, we profiled phosphorylation state of cellular proteins before and after growth factor stimulation (manuscript in preparation). We discovered that EGF induced the phosphorylation of cellular PTPN13 at both threonine 1336 and serine 1339 (Figure 3a). These two sites in PTPN13 were then individually or together mutated to alanine (PTPN13T1336A, PTPN13S1339A and PTPN13T1336A/ S1339A ) to prevent their phosphorylation after growth factor stimulation and to study whether phosphorylation of these two sites of PTPN13 has any effect on Her2
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PTPN13 mutations in human cancer promote Her2 phosphorylation Mutations in PTPN13 have been reported in clinical human tumors (Wang et al., 2004). To examine their impact on Her2 phosphorylation in cancer cells, we generated expression plasmids for two of these cancer mutants, PTPN13E2455D and PTPN13Y2260X (Figure 4a). Expression of either PTPN13E2455D or PTPN13Y2260X led to increased phosphorylation of cellular Her2 after growth factor induction (Figure 4b). Phosphatase activity assays showed that PTPN13E2455D retained around 50% of wild-type
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Figure 2 Increased expression of cellular PTPN13 inhibits Her2 phosphorylation, and Her2 forms a stable complex with a phosphatase inactive form of PTPN13. (a) HEK293 cells were transfected with empty vector (EV), expression plasmids encoding Flag-tagged wild-type PTPN13 (WT) or its catalytically inactive mutant PTPN13D2359A (DA). Forty-eight hours post transfection, cells were lysed and the phosphatase activities of WT and DA were assessed as described in Materials and methods. Relative phosphatase activity in these cells was normalized using WT transfected cells whose value was taken as 100%. (b) HEK293 cells were transfected with expression plasmids encoding WT or DA. Mocktransfected cells and EV transfected cells were used as controls. Forty-eight hours post transfection, cells were serum starved overnight. The cells were then treated with EGF (10 ng ml 1) for 10 min and the levels of phospho-Her2 (P-Her2), Her2, the expression of Flag-tagged proteins and GAPDH in these cells were analysed by western blot. (c) HEK293 cells were cotransfected with an expression plasmid encoding Her2 along with plasmid encoding WT, DA or Flag-tagged MDC1 (mediator of DNA damage checkpoint 1) as a control. Forty-eight hours post transfection, cells were serum starved overnight followed by EGF (10 ng ml 1) stimulation for 10 min. Cell lysates were then prepared and immunoprecipitation were performed with anti-Flag antibody. The protein levels of Her2 in these immunocomplexes were detected by western blot.
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phosphorylation (Figure 3b). Following EGF stimulation, expression of PTPN13T1336A or PTPN13T1336A/S1339A led to elevated Her2 phosphorylation while expression of PTPN13S1339A has an effect similar to that of wild-type PTPN13 (Figure 3c). Phosphatase activity assay showed that PTPN13T1336A and PTPN13T1336A/S1339A retained only around 35 and 25% of its wild-type activity, respectively, while PTPN13S1339A retained about 80% activity (Figure 3d). These data indicate that growth factorinduced phosphorylation of PTPN13 is important for its phosphatase activity and for deactivating Her2.
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Figure 3 The growth factor-mediated phosphorylation of PTPN13 is involved in the dephosphorylation of Her2 by PTPN13. (a) The mass spectrometry diagram of phosphorylation of PTPN13T1336 and PTPN13S1339 after EGF stimulation. The lower (indicated by — 4) and the upper lines (indicated by -) in the graphs show the phosphopeptides (sequences indicated at the bottom of graphs) detected by mass spectrometry before and after EGF stimulation, respectively. (b) The linear diagram of function domains and the positions of T1336A and S1339A mutation sites in PTPN13. (c) HEK293 cells were transfected with expression plasmids encoding wild-type PTPN13 (WT), PTPN13T1336A (TA), PTPN13S1339A (SA) or PTPN13T1336A/S1339A (TSA). Forty-eight hours post transfection, the cells were serum starved and then treated with EGF (10 ng ml 1) for 10 min before harvesting. The levels of phospho-Her2 (P-Her2), Her2, the expression of Flag-tagged proteins and GAPDH in these cells were examined by western blot. (d) HEK293 cells were transfected with empty vector (EV) or expression plamids encoding WT, TA, SA or TSA. 48 h post transfection, cells were collected and phosphatase activities of EV, WT, TA, SA and TSA were assessed as described in Materials and methods. Relative phosphatase activity in these cells was normalized using WT transfected cells whose value was taken as 100%. Oncogene
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PTPN13 knockdown or PTPN13 cancer mutants promote the invasiveness of Her2-overexpressing cancer cells Her2 overexpression is detected in about 30% of human breast and ovarian cancers and is a marker of poor prognosis for cancer patients (Hayes and Thor, 2002). As PTPN13 inhibited Her2 signaling, we tested whether PTPN13 has a role in regulating malignant properties of Her2-overexpressing tumor cells. PTPN13 expression was silenced in a Her2-overexpressing ovarian carcinoma cell line, SKOV3, by infecting it with recombinant lentivirus carrying shRNA against PTPN13 (shPTPN13). In PTPN13
knockdown SKOV3 cells (SKOV3-shN13), Her2 phosphorylation induced by growth factor was increased compared to parental SKOV3 and SKOV3 infected with virus carrying empty vector (SKOV3-shEV) (Figure 5a). There was no difference in cell proliferation and migration rate between SKOV3-shN13 and SKOV3-shEV cells (data not shown). However, the invasive potential of SKOV3shN13 was increased by about two fold compared with that of parental SKOV3 and SKOV3-shEV (Figures 5b and c). We further tested whether PTPN13 mutations in human cancer have a similar effect in promoting tumor cell invasiveness. We found that SKOV3 cells expressing PTPN13 cancer mutants, Y2260X or E2455D, promoted the SKOV3 invasion by about 50 and 25% compared to mock- or empty vector-transfected SKOV3 cells (Figure 5d). The increase of invasion in SKOV3-shN13 cells is mostly derived from upregulated Her2 signaling since Her2 knockdown in SKOV3-shN13 cells by siRNA
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PTPN13 activity and PTPN13Y2260X lost almost all its phosphatase activity (Figure 4c). We also found that cancer mutant PTPN13Y2260X, but not PTPN13E2455D, stably associated with cellular Her2 protein (Figure 4d). These results suggested that the dysfunctional PTPN13 cancer mutants can upregulate Her2 malignant signaling by decreasing their phosphatase activity and/or by stably interacting with the Her2 signaling domain and consequently blocking the accessibility of other functional cellular phosphatases to Her2.
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Figure 4 PTPN13 mutations in human cancer promote Her2 phosphorylation. (a) A linear diagram showing the locations of cancer mutations Y2260X (X, stop codon) and E2455D in PTPN13. (b) HEK293 cells were transfected with empty vector (EV) or expression plasmids encoding wild-type PTPN13 (WT), PTPN13E2455D (ED), or PTPN13Y2260X (YX). Forty-eight hours post transfection, these cells were serum starved overnight. Then the cells were stimulated with EGF (10 ng ml 1) for 10 min and the levels of phospho-Her2 (P-Her2), Her2, Flag-tagged proteins and GAPDH in these cells were examined by western blot. (c) HEK293 cells were transfected with EV or expression plasmids encoding WT, ED or YX. Forty-eight hours post transfection, phosphatase activity was assessed as described in Materials and methods. Relative phosphatase activity in these cells was normalized using WT transfected cells whose value was taken as 100%. (d) HEK293 cells were cotransfected with expression plasmids encoding Her2 along with plasmids encoding WT, ED or YX. Forty-eight hours post transfection, these cells were serum starved overnight. Subsequently, the cells were stimulated by EGF (10 ng ml 1) for 10 min, cell lysates were prepared and used for immunoprecipitation with anti-Flag antibody. The protein levels of Her2 in the immunocomplexes were detected by western blot as indicated. Oncogene
Figure 5 PTPN13 knockdown or PTPN13 cancer mutants promote the invasiveness of Her2-overexpressing cancer cells. (a) Stable SKOV3 cell line carrying empty vector (shEV) or vector expressing shRNA against PTPN13 (shPTPN13) was stimulated by 10 ng ml 1 EGF for 15 min. Parental cell line or cell line carrying shEV were used as controls. These cells were collected and the protein levels of phospho-Her2 (P-Her2), Her2, PTPN13 and GAPDH were detected by western blot as indicated. (b) Invasive potential of parental, shEV and shPTPN13 cells were analysed as described in Materials and methods. The relative cell invasion in these cells was normalized using parental cells whose value was taken as 1. (c) Representative images of invaded cells of parental, shEV and shPTPN13 as described in (b). (d) SKOV3 cells were transfected with empty vector (EV) or with expression plasmids encoding PTPN13E2455D (ED) or PTPN13Y2260X (YX). Forty-eight hours post transfection, the invasive capacity of the cells was analysed and the relative cell invasion in these cells was normalized using mock-transfected cells whose value was taken as 1. The level of P-Her2 and Her2 was detected after 48 h transfection and overnight starvation followed by stimulation with 10 ng ml 1 EGF for 15 min.
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reduced their invasive potential to the level of control SKOV3-shEV cells (Figure 6). We also tested the effect of PTPN13 knockdown on Her2-overexpressing breast cancer cell line BT474. Similar to HeLa and SKOV3, PTPN13 knockdown lead to increased Her2 phosphorylation and cell invasiveness of BT474 (Supplementary Figure S3). As tumor cell invasiveness is directly linked to tumor metastasis, these data imply that PTPN13 may have a role in inhibiting the invasion and metastasis of Her2-overexpressing tumor cells (Figure 7).
Discussion
Relative cell invasion
Overactive Her2 is found in nearly 30% of mammary and ovarian cancers and is associated with a poor clinical prognosis (Hayes and Thor, 2002). Consequently, since Her2 is an important molecular target for cancer therapy (Kim et al., 2004), understanding the regulatory mechanisms that control the malignant activity of Her2 in cells should help in devising new and more effective approaches to treat Her2-dependent cancers. Her2 oncogenic signaling is through kinase–mediated phosphorylation of its cytosolic signaling domain. Thus, dephosphorylation of the Her2 signaling domain by phosphatase should confine the magnitude and duration of Her2 oncogenic signaling after its activation in cells. To date, two nonreceptor phosphatases (BDP1/PTPN18 (Gensler et al., 2004) and PTP1B/ PTPN1 (Bentires-Alj and Neel, 2007; Julien et al., 2007)) have been shown to be involved or implicated in regulation of Her2 activity and its downstream signaling. BDP1/ PTPN18 inhibits Her2 signaling by inhibiting Her2 phosphorylation (Gensler et al., 2004). Conversely, PTP1B positively regulates Her2 signaling since PTP1B deficiency postponed Her2-induced mammary tumorigenesis and protected from lung metastasis (Bentires-Alj and Neel,
2 1.6
2007; Julien et al., 2007). Herein, we described a third phosphatase, PTPN13, which blocked Her2 activity by dephosphorylating the Her2 cytoplasmic domain (Figure 7). We found knockdown of PTPN13 by siRNA significantly increased Her2 phosphorylation, while increased expression of PTPN13 inhibited Her2 activity. Similar to BDP1 and PTP1B, PTPN13 also belongs to the family of nonreceptor phosphatases. However, besides the phosphatase catalytic domain, PTPN13 also contains one FERM domain and five PDZ domains, which may contribute to protein or substrate binding specificity (Erdmann, 2003; Tonks, 2006). Intriguingly, we found that growth factor induced the phosphorylation of PTPN13 at two sites, which are important for phosphatase activity of PTPN13 and consequent inhibition of Her2 activity. These results indicate the existence of a growth factor-induced negative feedback mechanism of Her2 signaling through phosphatase PTPN13 (Figure 7). Both decreased expression and mutations of PTPN13 have been observed in human tumors (Inazawa et al., 1996; Wang et al., 2004; Yeh et al., 2006). It has been reported that PTPN13 is located on chromosome 4q21.3, a region frequently deleted in liver and ovarian cancers (Inazawa et al., 1996). Lower expression levels of PTPN13 contribute to higher proliferation rate of certain cancer cell lines (Yeh et al., 2006), while no biochemical or cellular phenotype has been attributed to the mutations of PTPN13 found in human tumors (Wang et al., 2004). Herein, we demonstrated that expression of two of the cancer mutants of PTPN13, PTPN13E2455D and PTPN13Y2260X, individually led to enhancement of Her2 activity. In addition, similar to the catalytically inactive mutant PTPN13D2359A described in Figure 2, the cancer mutant with little phosphatase activity, PTPN13Y2260X was also able to bind Her2 protein with much higher persistent affinity than wildtype PTPN13. This suggests that nonfunctional PTPN13 mutation(s) in cancer may promote Her2dependent malignancy not only by their inactivity but also by binding to Her2 and preventing the entry of other phosphatase(s) or molecules that downmodulate
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Figure 6 Increased invasion caused by PTPN13 knockdown is primarily due to Her2 signaling. Stable SKOV3 cell line carrying empty vector (shEV) or vector expressing shRNA against PTPN13 (shN13) was mock-transfected or transfected with siRNA against Her2 (siHer2) as indicated. After 3 days, the invasive potential of mock or Her2 siRNA transfected cells were analysed as described in Materials and methods. The relative cell invasion in these cells was normalized using mock-transfected shEV cells whose value was taken as 1.
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Figure 7 Schematic illustration of mechanisms of action of PTPN13 on Her2 malignant activity. Oncogene
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the activity of Her2. Importantly, expression of these two cancer mutants or knockdown of cellular PTPN13 increased cell invasiveness of Her2-overactive tumor cells. As cell invasiveness is closely connected to the metastatic potential of tumor, these data suggest that PTPN13 negatively regulates Her2 activity and consequently may inhibit metastasis of the Her2-overexpressing tumor (Figure 7). Therefore, cellular PTPN13 likely possesses tumor suppressive activity, which has also been suggested by reports from other labs (Bompard et al., 2002; Ostman et al., 2006; Yeh et al., 2006). Her2/ErbB2 belongs to the family of ErbB/EGF receptors, which includes three other members, Her1/ EGFR/ErbB1, Her3/ErbB3 and Her4/ErbB4. Upon growth factor stimulation, these receptors homodimerize or heterodimerize leading to autophosphorylation and subsequent downstream effector activation (Engelman and Cantley, 2006). Due to the similar architecture among the ErbB receptors, a single phosphatase may be able to modulate more than one of these receptors. However, to date, no phosphatase has been shown to directly dephosphorylate both Her1 and Her2. PTP1B was reported to regulate Her1 signaling since cells deficient in PTP1B exhibited increased and sustained phosphorylation of Her1 after growth factor treatment (Haj et al., 2003). This phosphatase was also involved in regulation of Her2 signaling, likely by functioning on downstream pathways (Bentires-Alj and Neel, 2007; Julien et al., 2007). BDP1/PTPN18 can only regulate one ErbB receptor as increased expression of this phosphatase-inhibited growth factor induced activation of Her2 but not that of closely related Her1 (Gensler et al., 2004). Interestingly, similar to BDP1, PTPN13 knockdown led to minimal, if any, change of Her1 phosphorylation level after growth factor treatment (Supplementary Figure S1A). In summary, our data showed that PTPN13 negatively regulated Her2 activity and inhibited invasiveness of Her2-overexpressing tumor cells. In addition, we discovered that growth factor induced the phosphorylation of PTPN13, which was important for the ability of PTPN13 to inhibit the Her2 malignant signaling indicating a growth factor-induced negative feedback mechanism of Her2 activity through PTPN13. PTPN13 mutations in human tumors escalated Her2 activity and the invasive potential of Her2-deregulated cancer cells suggesting that cellular PTPN13 has a role in containing the Her2 oncogenic signaling and inhibiting the invasive and metastatic potential of tumors bearing overactive Her2. Finally, the identification of PTPN13 as a direct regulator of Her2 activity provides an important clue for designing novel approaches to slow down or stop tumor invasiveness and metastasis of Her2-dependent cancers.
Materials and methods Cell culture The human cell lines HeLa, HEK293 and SKOV3 were obtained from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Oncogene
Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% L-glutamine (Invitrogen, Carlsbad, CA, USA). siRNA library screen The phosphatase siRNA library and transfection reagent were purchased from Dharmacon (Lafayette, CO, USA). The siRNA against each gene was a pool composed of four individual siRNA duplexes. siRNA transfection into HeLa cells was conducted following the manufacturer’s protocol. Cells were transfected with siRNA for 2 days and then serum starved for 1 day before harvest. After stimulated for 15 min with 10 ng ml 1 EGF, cells were lysed in RIPA buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 mg ml 1 PMSF and 1 mM sodium orthovanadate). Western blot was performed to screen for changes in Her2 phosphorylation. DNA constructs and transfection The lentiviral shRNA plasmid against PTPN13 was purchased from Open Biosystems (Huntsville, AL, USA). The SKOV3 cell lines carrying PTPN13 shRNA or the empty vector were established according to the manufacturer’s protocol. The Flagtagged PTPN13 expression plasmid was a generous gift from Dr Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala University, Sweden). PTPN13 mutations were constructed using Quikchange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The Flag-tagged MDC1 (mediator of DNA damage checkpoint 1) expression plasmid was provided by Dr Maoyi Lai (The Scripps Research Institute, La Jolla, CA, USA). HEK293 cells were transfected using a calcium phosphate method modified from the protocol provided by Open Biosystems. On the other hand, SKOV3 cells were transfected using GenJet DNA in vitro transfection reagent (SignaGen Laboratories, Gaithersburg, MD, USA) following the manufacturer’s recommended protocol. Antibodies, western blot and immunoprecipitation Anti-Flag antibody was purchased from Stratagene. Antiphospho-Her2 (Tyr1248), anti-Her2, anti-phospho-Akt (Ser473) and anti-Stat3 (Tyr705) antibodies were from Upstate (Lake Placid, NY, USA). Anti-phospho-Erk1/2 (Thr202/ Tyr204) antibody was from Cell Signaling (Boston, MA, USA). Anti-GAPDH antibody was from Calbiochem (San Diego, CA, USA). Anti-BMK1 antibody was previously described (Kato et al., 1997). Anti-PTPN13 antibody, antirabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). For western blot, cells were lysed in RIPA buffer as described above. For immunoprecipitation, cells were lysed in 50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 10% glycerin, 1% Triton X-100, 100 mg ml 1 PMSF and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Samples were loaded onto 6 or 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and examined by western blot. Phosphoproteomic analysis Phosphoproteomic analysis of the EGF pathway was carried out as detailed elsewhere (Cantin GT, Yi W, Lu B, Park SK, Xu T, Lee JD and Yates JR; manuscript in preparation). Briefly, HeLa cells were grown separately in heavy and light SILAC D-MEM media (Invitrogen), and one culture was treated with EGF. Protein lysates were prepared from both cultures, mixed 1:1 and subjected to two rounds of immobilized metal affinity chromatography (IMAC). Peptides from the final
PTPN13 negatively regulates Her2 activity J-H Zhu et al
2531 IMAC eluate were subjected to MudPIT (Washburn et al., 2001) and relative quantification analysis (Cantin et al., 2006). Invasion assay Cell invasion was analysed as described by Hauck et al. (2002) with slight modification. Briefly, Matrigel (Sigma, Saint Louis, MI, USA) was thawed at 4 1C overnight, and diluted to around 3 mg ml 1 with cold DMEM. Millicell plate inserts (12 mm diameter with 8.0 mM pores, Millipore, Billerica, MA, USA) were coated with 100 ml diluted Matrigel and incubated at 37 1C 4–5 h for gelling. Cells were harvested by Trypsin/ EDTA, washed twice with DMEM, and resuspended in DMEM at a density of 106 per ml. Lower chambers were filled with 600 ml warm DMEM containing 10% FBS as the attractant, and the Matrigel-coated upper chambers were filled with 100 ml of the cell suspension. After 24 h incubation at 37 1C, cells were fixed for 30 min in 5% glutaraldehyde prepared in phosphate-buffer saline at room temperature, and stained 30 min in 0.1% crystal violet and 2% ethanol. The numbers of invasive cells were measured by counting three fields per chamber. Mean values were obtained from at least three separate experiments.
Phosphatase activity assay The method of phosphatase activity assay was modified from Xu et al. (2005) and the protocol of Upstate. Briefly, Flag-tagged protein was immunoprecipitated using anti-Flag antibody. Bovine serum albumin (5 ml, 5 mg ml 1) was added to the immunocomplex and total volume was brought to 80 ml with phosphatase assay buffer (25 mM HEPES, pH 7.2, 50 mM NaCl, 5 mM dithiothreitol, 2.5 mM EDTA). Thereafter, 120 ml of p-nitrophenyl phosphate (4 mg ml 1) freshly prepared in the assay buffer was added into the mixture to start the reaction. After 15 min at 37 1C, the reaction was stopped by adding 20 ml of 13% K2HPO4. Then 60 ml of the mixture was transferred to a 96-well plate, and hydrolysis of p-nitrophenyl phosphate was measured spectrometrically at 405 nm. Activity was expressed as a relative value of wild-type PTPN13 activity, which was set to 100%. Mean values were obtained from three individual experiments. Acknowledgements This work was supported by funds from National Cancer Institute (CA079871 and CA114059), California Tobacco-Related Disease (15RT-0104) and Department of Defense BCRP (BC031105).
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc). Oncogene
