NIH Public Access Author Manuscript Oncogene. Author manuscript; available in PMC 2012 August 25.
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Published in final edited form as: Oncogene. 2011 August 25; 30(34): 3705–3715. doi:10.1038/onc.2011.90.
Mutant K-Ras increases GSK-3β gene expression via an ETSp300 transcriptional complex in pancreatic cancer J-S Zhang1,5, A Koenig1,2,5, A Harrison1, AV Ugolkov1,4, ME Fernandez-Zapico1, FJ Couch3, and DD Billadeau1 1Department of Immunology and Division of Oncology Research, Schulze Center for Novel Therapeutics, College of Medicine, Mayo Clinic, Rochester, MN, USA 2Department
of Gastroenterology and Endocrinology, Philipps-University of Marburg, Marburg,
Germany 3Division
of Experimental Pathology and Laboratory Medicine, Mayo Clinic, Rochester, MN, USA
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Abstract Glycogen synthase kinase-3 beta (GSK-3β) is overexpressed in a number of human malignancies and has been shown to contribute to tumor cell proliferation and survival. Although regulation of GSK-3β activity has been extensively studied, the mechanisms governing GSK-3β gene expression are still unknown. Using pancreatic cancer as a model, we find that constitutively active Ras signaling increases GSK-3β gene expression via the canonical mitogen-activated protein kinase signaling pathway. Analysis of the mechanism revealed that K-Ras regulates the expression of this kinase through two highly conserved E-twenty six (ETS) binding elements within the proximal region. Furthermore, we demonstrate that mutant K-Ras enhances ETS2 loading onto the promoter, and ETS requires its transcriptional activity to increase GSK-3β gene transcription in pancreatic cancer cells. Lastly, we show that ETS2 cooperates with p300 histone acetyltransferase to remodel chromatin and promote GSK-3β expression. Taken together, these results provide a general mechanism for increased expression of GSK-3β in pancreatic cancer and perhaps other cancers, where Ras signaling is deregulated.
Keywords
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GSK-3β; K-Ras; MAPK; ETS; pancreatic cancer
Introduction The glycogen synthase kinase-3β (GSK-3β) is a ubiquitously expressed serine/threonine kinase, which participates in various cellular functions such as metabolism, proliferation, differentiation and apoptosis (Luo, 2009). The role of GSK-3β in cancer is somewhat enigmatic. In part, this is attributed to its function within the canonical Wnt signaling cascade, where it regulates the levels of the oncoprotein β-catenin, thus functioning as a © 2011 Macmillan Publishers Limited All rights reserved Correspondence: Dr J-S Zhang or Professor DD Billadeau, Division of Oncology Research, 200 First Street SW, Rochester, MN 55905, USA.
[email protected] or
[email protected]. 4Current address: Department of Pathology, Northwestern University, Chicago, IL, USA. 5These authors contributed equally to this work. Conflict of interest The authors declare no conflict of interest. Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
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putative tumor suppressor (Rubinfeld et al., 1996; Polakis, 2000). However, it cannot be overlooked that numerous reports have demonstrated increased GSK-3β protein expression in human malignancies including pancreatic, renal, colon, ovarian cancer and glioblastoma (Ougolkov et al., 2005; Shakoori et al., 2005; Cao et al., 2006; Kotliarova et al., 2008; Bilim et al., 2009). In particular, in pancreatic carcinogenesis, GSK-3β protein becomes overexpressed progressively from early pancreatic intraepithelial neoplasia to PDA (pancreatic ductal adenocarcinoma) (Ougolkov et al., 2006), suggesting that early genetic changes in pancreatic intraepithelial neoplasia lesions could influence GSK-3β protein expression. Importantly, increased expression of GSK-3β and its kinase activity are responsible for enhanced cell proliferation, survival and chemo-resistance in PDA (Ougolkov et al., 2005; Billadeau, 2007). Significantly, the molecular mechanism driving this overexpression is still unknown.
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PDA is the most common pancreatic tumor and among one of the most aggressive and devastating human malignancies. Several common genetic alterations have been identified, including activating K-Ras mutations or the loss of tumor suppressor proteins like DPC4/ Smad4, p53, BRCA2 or p16, as cornerstone in pancreatic carcinogenesis (Hezel et al., 2006; Maitra et al., 2006). Activating mutations in the K-Ras proto-oncogene are widely accepted as the most frequent and among the earliest genetic alterations in PDA development (Deramaudt and Rustgi, 2005). A prominent nuclear effector of Ras signaling is the Etwenty six (ETS)/Ets-LiKe (ELK) family of transcription factors. These proteins participate in embryonic development, maintenance of adult tissues and have been shown to have an important role in various human malignancies (Wasylyk et al., 1998; Jedlicka and GutierrezHartmann 2008). Interestingly, in addition to the Ewing sarcoma (EWS)–ETS fusion found in Ewing’s sarcoma (Arvand and Denny, 2001; Janknecht, 2005) and the more recently identified TMPRSS2-ETS in prostate cancer (Narod et al., 2008; Clark and Cooper, 2009), aberrant expression of ETS proteins has been reported in PDA and other gastrointestinal malignancies (Ito et al., 1998, 2002, 2004; Wai et al., 2006; Gutierrez-Hartmann et al., 2007). Beside the discovery of SRE and c-Fos as ELK-1-responsive target genes (Li et al., 2003), recent work elucidated the role of ELK-1 in cooperation with NFATc2, in regulation of c-Myc expression (Koenig et al., 2010). However, despite their widespread expression and involvement in multiple cellular processes, very little is known about direct target genes of ETS family members, especially in carcinogenesis.
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Although the regulation of GSK-3β function and activity has been extensively studied (Jope and Johnson, 2004), the transcriptional regulation of GSK-3β leading to its aberrant expression in cancer cells is unknown. A previous study has mapped the transcription start within the GSK-3β promoter and identified a 2.5 kb fragment, which showed promoter activity in several cell lines (Lau et al., 1999a). In the current study, we characterized the regulation of GSK-3β transcription in pancreatic cancer cells and identified an active Ras– mitogen-activated protein kinase (MAPK)–ETS2–p300 cascade leading to GSK-3β overexpression in pancreatic cancer cells.
Results Mutant Ras regulates GSK-3β transcription via MAPK signaling We have previously shown that GSK-3β protein becomes overexpressed progressively from pancreatic intraepithelial neoplasia stages to PDA. Interestingly, we also observed high expression of GSK-3β in tumor cells derived from a murine, monogenetic-determined pancreatic tumor model expressing K-RasG12D driven by the pancreas specific Pdx-1 promoter (Figure 1a). Moreover, tumor cells obtained from these mice exhibit higher levels of GSK-3β than cells obtained from mice with an engineered homozygous loss of the p53 and BRCA-2 genes (Figure 1b), suggesting a correlation of K-Ras activity and GSK-3β Oncogene. Author manuscript; available in PMC 2012 August 25.
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expression. This assumption is strengthened by the fact that mutant K-Ras expressing cells are more sensitive to GSK-3 inhibition. As shown in Figure 1c increasing amounts of the GSK-3 inhibitor LY2064827 blocked tumor growth more efficiently in K-RasG12D cells, compared with cells containing wild type K-Ras. At a concentration of 2.5 μM, tumor growth inhibition ranges between 44% (24 h) and 64% (72 h) in K-RasG12D derived cells, whereas in p53−/−/BRCA2−/−-derived cells, the growth inhibition is only 27% (72 h) at maximum compared with dimethyl sulfoxide-treated controls (Figure 1d). Furthermore, overexpression of mutant K-Ras in p53−/−/BRCA2−/− cells increased growth of these tumor cells, which is almost reverted to the level of control-transfected cells by GSK-3 inhibition (Figure 1e). These results suggest a contribution of K-Ras activity to GSK-3β expression and GSK-3βmediated function in pancreatic cancer cells. Interestingly, we also observed a correlation of GSK-3β mRNA levels with K-RasG12D expression (Figure 2a), which prompted us to further characterize the transcriptional regulation of GSK-3β by K-Ras signaling in pancreatic cancer cells.
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To determine the influence of mutant K-Ras on GSK-3β expression and related promoter activity, we cloned the human GSK-3β promoter for further analysis. We tested both the 0.5 kb (−427/+66) and 2.5 kb (−2450/+66) promoter constructs in human pancreatic cancer cell lines. Interestingly, both constructs revealed similar activities in luciferase reporter assays in HupT3 (Figure 2b), as well as BxPC-3 and Panc 04.03 (data not shown), suggesting that indeed the regulatory elements mainly reside within this proximal region as assumed (Lau et al., 1999a). In agreement with the elevated GSK-3β expression in murine K-RasG12D expressing cells, depletion of K-Ras by short hairpin RNA (shRNA) (Supplementary Figure S1A), leads to both reduced GSK-3β promoter activity (Figure 2c) and a reduction of endogenous GSK-3β mRNA expression, when compared with control cells (Figure 2d).
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To identify specific signaling pathway(s) contributing to the regulation of GSK-3β promoter, we screened a panel of protein kinase inhibitors and found that inhibitors against MAPK (PD98059 and U0126), but not PI3K/mammalian target of rapamycin (wortmannin) or protein kinase C (G06976), could significantly reduce the activity of the (−427/+66) promoter construct, suggesting MAPK signaling as the major regulatory pathway on the GSK-3β promoter in pancreatic cancer cells (Figure 2e). Although all three kinase inhibitors affect cell growth and perturb the cell cycle (Supplementary Figure S1B, C), only mitogenactivate protein 2 kinase 1 inhibitors reduced the promoter activity (Figure 2e), suggesting a specific effect of MAPK signaling on GSK-3β promoter regulation. Consistent with the results of the luciferase reporter assays, we observed decreased levels of GSK-3β mRNA by quantitative real time PCR (RT–PCR) in HupT3 cells upon treatment with U0126 under serum free conditions (Figure 2f). The decrease in GSK-3β mRNA levels is recapitulated by a reduction in endogenous GSK-3β protein expression (Figure 2g), which drops to 20% of control at 48 h of U0126 treatment (Figure 2h). As activated Ras signaling is common in various human tumors, we determined the effect of each Ras isoform on the promoter activity. Interestingly, all three Ras isoforms are capable of further enhancing GSK-3β promoter activity in BxPC-3 cells, which contain wild type KRas (Figure 3a). Even in HupT3 cells harboring an activating K-Ras mutation, the GSK-3β promoter is still inducible by overexpression of either constitutively active H-Ras or K-Ras (Figure 3b). More importantly, treatment of HupT3 cells with the MEK1/2 inhibitors resulted in a reduction of promoter activity to approximately 60% (PD98059) or 40% (U0126), respectively, compared with dimethyl sulfoxide-treated cells, regardless of HRasG12V and/or K-RasG12D overexpression (Figure 3c). Additionally, neither constitutively active K-Ras nor H-Ras or their combination is able to induce GSK-3β promoter activity in the presence of U0126 in BxPC-3 cells (Figure 3d). Together, the data suggest that the MAPK pathway has a dominant role in mediating Ras-induced GSK-3β promoter activity.
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The observed Ras-dependent alterations of the GSK-3β promoter activity are revealed at the transcription level in that constitutively active K-Ras and H-Ras are able to increase GSK-3β mRNA based on quantitative RT–PCR, whereas treatment with U0126 abolished this effect (Figure 3e). Overexpression of active K-Ras or H-Ras in BxPC-3 cells increases GSK-3β protein expression by nearly two-fold, whereas treatment with U0126 prevents both ERK phosphorylation and Ras-induced GSK-3β expression as expected (Figure 3f). Taken together, these data indicate that a Ras–MAPK signaling pathway is involved in GSK-3β gene expression. ETS transcription factors mediate Ras/MAPK induction of GSK-3β promoter activity To identify the Ras/MAPK responsive elements within the GSK-3β promoter, we generated and tested a series of 5′ deletion constructs in reporter assays (Figure 4a). Deletion of the promoter sequence from the 5′ end results in a steady decrease of promoter activity in both cell lines, although HupT3 cells demonstrated higher basal activity (Figure 4b). However, all constructs could be stimulated by overexpression of constitutively active K-Ras (Figure 4c and data not shown). In contrast, the 3′-deletion construct lacking 157 bp downstream of the transcription start lost responsiveness to K-RasG12D (Figure 4c, Supplementary Figure S2B), suggesting that regulatory elements of the Ras/MAPK pathway are located within the proximal region (−165/+66).
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Several additional 5′-deletion constructs were made and tested in reporter assays and all of them demonstrate reduced activity, when MAPK signaling is blocked in cells with active KRas mutation (Figure 5a). Consistent with this result, all constructs could be further stimulated by expression of a constitutively active Ras isoform in BxPC-3 cells. However, the two longest constructs (−427/+66, −165/+66) revealed a nearly two-fold higher response compared with the shorter constructs (−103/+66, −53/+66) pointing to two separable regulatory regions – one within −165 to −103 and another within −103 to +66. A detailed sequence analysis of −165/+66 region revealed a number of potential binding sites for several transcription factors including AP1 and ETS (Figure 5c, Supplementary Figure S3D). As ETS proteins are well-known nuclear effectors of Ras/MAPK, the presence of three ETS binding sites is of particular interest. The three potential binding sites (GGAA) are located at −139 (AGGAAG), −97 (GGGAAG) and +18 (AGGAAGGAAGGAAG), relative to transcription start. The third ETS site contains a tandem repeat of the core sequence, adjacent to a putative AP1 site (shaded in green, Supplementary Figure S3D). To determine the role of these putative binding sites to Ras/MAPK signaling, we mutated each binding site alone and in combination in the context of the −165/+66 reporter construct (Figure 5c, Supplementary Figure S3D). These constructs were tested for their response to overexpression of H-RasG12V in reporter assays. Mutant A showed decreased responsiveness, mutant B had no effect, whereas combined mutation of A, B and C resulted in a nearly complete loss of Ras responsiveness, suggesting that the ETS A and C sites are crucial in the regulation of GSK-3β by RAS/MAPK signaling (Figure 5d). We further generated and tested the reporter constructs with 3′ deletions. Upon removal of the putative AP1/ETS composite site (−165/+14), the reporter activity was reduced about 50% in response to active Ras, whereas the other deletion containing an intact AP1/ETS composite site (−165/+37) revealed similar response as the wild type (−165/+66), suggesting the existence of a second regulatory element within the putative AP1/ETS composite site (Supplementary Figure S3A). Gel shift analysis confirmed the direct binding of several nuclear proteins to these sites (Supplementary Figure S3B), which are efficiently competed away by wild type cold probe and known ETS-binding oligos, but not their respective mutant oligos, suggesting that multiple ETS proteins may bind these elements (Supplementary Figure S3C).
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ETS proteins are known to have important roles in tumorigenesis and translocation, and/or overexpression of ETS proteins have been observed in many human malignancies (Jedlicka and Gutierrez-Hartmann, 2008). Indeed, we observed abundant expression of both ETS1 and ETS2, the predominant members of the ETS family in all tested pancreatic cancer cell lines (Figure 6a). Moreover, overexpression of either ETS1 or ETS2 proteins robustly activates the GSK-3β promoter (Figure 6b). However, mutations of the known MAPK phosphorylation sites in ETS1 (T38A) or ETS2 (T72A) disrupted their transactivation, indicating an importance of MAPK signaling in mediating their effect on the GSK-3β promoter (Figure 6b, Supplementary Figure S2C). Overexpression of an ETS protein containing only the DNA-binding domain (dn-ETS) abrogated the activity of all tested promoter constructs in a concentration dependent manner (Figure 6c, Supplementary Figure S2D). Furthermore, co-expression of ETS2 and K-RasG12D in BxPC-3 cells resulted in a dramatic synergistic effect with 45-fold increase over the control as compared with 3.6- or 21-fold with K-RasG12D or ETS2 alone (Figure 6d). Importantly, in the presence of dn-ETS, K-RasG12D fails to activate the promoter, suggesting an essential role of ETS proteins in Ras-activation of the GSK-3β promoter (Figure 6e). In fact, we find that both ETS2 and cJun can interact with GSK-3β promoter in pancreatic cancer cells harboring active K-Ras as demonstrated by chromatin immunoprecipitation (ChIP) (Supplementary Figure S2E). More importantly, overexpression of K-RasG12D in BxPC-3 cells leads to a 2.5-fold increase in binding of ETS2 to the GSK-3β proximal promoter region (Figure 6f), suggesting that the activation of the GSK-3β promoter by ETS overexpression or Ras-stimulation of the MAPK pathway modifies the chromatin landscape. P300 cooperates with ETS2 to remodel chromatin and activate GSK-3β promoter ETS proteins have been shown to recruit co-activators with histone acetylase activity to activate target gene transcription (Yang et al., 1998; Foulds et al., 2004). As expected, coexpression of the histone acetylase p300, together with ETS2 resulted in a dramatic synergistic increase in GSK-3β promoter activity of nearly 50-fold, compared with a 12.2fold and 8.1-fold increase with p300 or ETS2 alone (Figure 7a). Consistent with the effect of p300 expression on GSK-3β promoter activity, the histone deacetylase inhibitor trichostatin A (TSA) significantly increased GSK-3β promoter activity (Figure 7b). Furthermore, ETS2 overexpression combined with TSA treatment led to more than 50-fold increase in reporter activity (Figure 7b). Simultaneous expression of K-RasG12D, ETS2 and treatment with TSA increased the activity of the GSK-3β promoter by 189-fold over the control in BxPC-3 cells, whereas inhibition of MAPK signaling with U0126 or co-expression of dn-ETS and KRasG12D markedly alleviated the activation of the GSK-3β promoter by TSA, reducing the activity to 27 or 6% of maximal stimulation (Figure 7c).
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The binding of ETS2 to the proximal GSK-3β promoter and subsequent change of the chromatin structure following Ras activation was further evaluated by ChIP. Although a significant binding of ETS2 to the promoter is observed in Panc 04.03 cells, treatment of the cells with U0126 leads to dissociation of ETS2 from the GSK-3β promoter, whereas the binding of c-Jun remains relatively unchanged (Figure 7d, top panel). U0126 treatment also resulted in reduced acetylation of histone 3 at lysine 14, a known activation mark and a substrate of histone acetylase p300, as well as reduced loading of RNA polymerase II, consistent with reduced transcriptional activity of GSK-3β gene (Figure 7d, lower panel). Following, we wanted to determine if K-Ras-dependent ETS2/p300 transactivation of GSK-3β promoter observed in reporter and ChIP assays can be fully recapitulated by the change of the endogenous GSK-3β protein expression. Indeed, overexpression of KRasG12D, ETS2 or inhibition of histone deacetylation with TSA each leads to an increase in GSK-3β expression in the BxPC-3 cells, whereas disruption of the K-Ras/MAPK/ETS/p300 activation cascade by dn-ETS prevents these effects (Figure 7e).
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Because of the dramatic synergy between mutant K-Ras and ETS2 in the activation of GSK-3β expression, we determined the functional relevance of this regulation in K-Rasmediated tumor growth. Concomitant over-expression of mutant K-Ras and ETS2 in BXPC3 cells, which contain wild type K-Ras, enhanced cell growth compared with mutant K-Ras alone. Importantly, this growth stimulatory effect was largely abolished upon GSK-3 inhibition. These results underscore an important role for the MAPK–ETS2–GSK–3β regulatory circuit in mutant K-Ras-mediated cell growth in pancreatic cancer (Figure 7f).
Discussion
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Although ubiquitously expressed, the levels of GSK-3β expression vary widely in different cell types, as well as in tumor tissues (Lau et al., 1999b; Hollenhorst et al., 2004). Moreover, there is increasing evidence that aberrant expression of GSK-3β contributes to cell proliferation/survival in several human malignancies including pancreatic, colon, ovarian, prostate and renal cell cancer (Shakoori et al., 2005; Cao et al., 2006; Ougolkov et al., 2006; Bilim et al., 2009; Li et al., 2009b). In particular, in pancreatic carcinogenesis we have already shown that inhibition or genetic depletion of GSK-3β leads to an increase in apoptosis due to diminished nuclear factor-κB activity (Ougolkov et al., 2005). However, the underlying mechanism(s) leading to overexpression of GSK-3β in human cancer remain unknown. We have previously reported the overexpression of GSK-3β in pre-neoplastic pancreatic intraepithelial neoplasia lesions to PDA resembling the mutational profile of KRas in pancreatic carcinogenesis, suggesting a possible role for this oncogene in the observed overexpression (Ougolkov et al., 2006). Consistent with this notion, in the present study, we provide evidence that GSK-3β mRNA/protein expression is associated with transgenic expression of K-RasG12D in PDA mouse cell lines. Furthermore, we identified two cis-regulatory elements within the GSK-3β promoter that bound ETS transcription factors. Finally, we demonstrate that Ras-MAPK signaling promotes ETS binding through histone acetylation that is likely mediated by p300. Thus, our observation of Ras–MAPK regulation of the GSK-3β promoter provides a general mechanism for the overexpression of GSK-3β, as this signaling pathway is activated in a majority of human cancers.
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As the founding members of the ETS family, both ETS1 and ETS2 have been reported to be overexpressed in PDA (Ito et al., 1998, 2002). Consistent with this, we also detected abundant ETS1 and ETS2 expression in pancreatic cancer cell lines compared with the human pancreatic ductal epithelial-immortalized cell line, thus supporting the relevance of K-Ras–MAPK–ETS cascade in the activation of GSK-3β expression in PDA. In fact, overexpression of dn-ETS not only reduced the basal GSK-3β transcription with mutant KRas, but also abolished the Ras-induced superactivation of GSK-3β in pancreatic cancer cells with wild type Ras. Moreover, mutation of the two putative ETS binding sites is sufficient to abrogate the effect of Ras-induced response. Taken together, these data suggest that ETS proteins are the major, if not the only, transcription factors mediating the Ras responsiveness of the GSK-3β promoter. It is worth noting that we employed a dominant negative form of ETS instead of small interfering/shRNA to block the activity of endogenous ETS proteins. The dn-ETS2 construct encoding the highly conserved DNA-binding (ETS) domain, but lacking the transactivation domain, has been shown to be capable of localizing to the cell nucleus and competing with wild type proteins binding to the cis-regulatory sequences. This approach has been widely used in numerous in vitro and in vivo studies including the PDA (Lefter et al., 2009; Li et al., 2009a). Given that ETS proteins recognize the similar core element GGA(A/T), especially ETS1 and ETS2 sharing almost identical ETS domains, the dn-ETS is advantageous over small interfering/shRNA in simultaneously suppressing the activity of ETS1 and ETS2 and likely other ETS, such as ELK and Pea3 (ETV4) (Hollenhorst et al.,
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2007). Both latter genes are also Ras/MAPK effectors expressed in PDA and have been shown to transactivate the MUC4 promoter aberrantly activated in PDA (Fauquette et al., 2005), or participate in serum-induced NFAT-mediated transactivation of Myc oncogene in pancreatic cancer cells (Koenig et al., 2010). However, the expression levels of both Pea3 and ELK are much lower in pancreas than ETS1/ETS2 (Kobberup et al., 2007; Zhang et al., unpublished observation). ELK overexpression showed much less pronounced transactivity of GSK-3β as compared with ETS2 in reporter assays (data not shown). These data suggest that ETS1 and ETS2 are the major ETS members mediating Ras/MAPK activation of GSK-3β in PDA.
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The effect of Ras/MAPK phosphorylation on ETS proteins differs among family members ranging from altered subcellular localization to a change in DNA binding affinity or transactivation/repression potentials (Tootle and Rebay, 2005). ETS1 and ETS2 function as transcriptional activators through recruitment of the general co-activators CBP/p300 (Yang et al., 1998; Jayaraman et al., 1999). Ras/MAPK signaling via phosphorylation of a highly conserved residue in ETS1 (T38) and ETS2 (T72), stimulate their transactivity through enhanced interaction with the histone acetylase CBP/p300 (Yang et al., 1996; Foulds et al., 2004; Nelson et al., 2010). In support of this notion, we could show that active Ras/MAPK signaling modulates the chromatin landscape of the GSK-3β promoter as reflected in acetylation of H3K14, a major site for p300-mediated histone acetylation correlated with transcriptional activation (Daujat et al., 2002). Together, these data indicate that K-Ras/ MAPK/ETS2/p300 cascade is not only operating in PDA, but appears to have a major role in driving GSK-3β overexpression. Our findings may have broader implications considering that nearly 30% of all human tumors harbor activating mutations in one of the three Ras genes or Raf leading to MAPK activation (Downward, 2003; Kim and Choi, 2010), which based on our results, all have the potential to activate GSK-3β transcription. It will be interesting to determine if the same cascade functions in other tumors with GSK-3β overexpression. In summary, our results have linked GSK-3β overexpression to K-Ras activation, one of the most common and earliest genetic alterations in PDA. We provide evidence that GSK-3β is a bona fide transcriptional target of ETS onco-proteins. We propose that Ras–MAPK– ETS2–p300 membrane to nucleus signaling is the major operating machinery, leading to activation of GSK-3β promoter and its overexpression in PDA. Mechanistically, active Ras– MAPK signaling, through enhanced recruitment of p300 to create a permissive chromatin environment, facilitates ETS2 loading to the GSK-3β promoter and its transactivation. Thus, this study provides mechanistic insight of GSK-3β overexpression in PDA, which may also have implications in other GSK-3β overexpressing tumors.
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Materials and methods Cells, transfections and treatment The mouse PDA cell lines expressing K-RasG12D were a gift from Dr Pinku Mukherjee. The mouse PDA cell line lacking p53 and BRCA2 was recently described (Rowley et al., 2011). All other pancreatic cancer cell lines were obtained from American Type Culture Collection. MAPK inhibitor PD98059 and PI3 kinase inhibitor wortmannin were purchased from Cell Signaling Technology (Danvers, MA, USA). The MAPK inhibitor U0126 and protein kinase C inhibitor G06976 were obtained from Promega (Madison, WI, USA) and Calbiochem (San Diego, CA, USA), respectively. The GSK-3 specific kinase inhibitor (LY2064827) was obtained from Eli Lilly (Indianapolis, IN, USA). Transfections were performed with either electroporation or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
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Plasmids
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The DNA sequence corresponding to −2250/+66 of the human GSK-3β promoter region (Lau et al., 1999a) was PCR amplified from normal human genomic DNA, and additional deletions were made and inserted into pGL3 basic vector (Promega). The EGFP-Ras cDNAexpression plasmids were kindly provided by Dr K Giehl. All other Ras expression plasmids were generated by RT–PCR and inserted into mammalian expression vectors with an Nterminal tag as specified. The expression vectors for ETS1/2 and p300 were kindly provided by Dr Ralph Janknecht. The cDNA sequence encoding the DNA-binding domain (amino acids 330-469) of hETS2 was subcloned with an N-terminal Flag, which was used as dominant negative form of ETS (dn-ETS). Site-directed mutagenesis of promoter and expression constructs were performed using Quickchange mutagenesis kit (Stratagene, Santa Clara, CA, USA). The shRNA expression constructs targeting K-Ras were cloned into pFRT vector at BglII/HindIII (sequences in Supplementary Table 2). All cDNA and shRNA expression plasmids were verified by direct sequencing at the Mayo Molecular Biology Core Facility. Immunohistochemistry and protein analysis
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Immunohistochemical analysis of pancreatic tumors explanted from the transgenic mice (KRasG12D) was performed as described previously (Ougolkov et al., 2006). The slides were evaluated by standard light microscopy (Carl Zeiss, Thornwood, NY, USA) at 200-fold magnification. For protein analysis, cells were lysed with radioimmunoprecipitation assay buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mmol/l sodium chloride, 50 mmol/l Tris/HCl (pH 7.2), 10 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l ethylene glycol acetic acid) and immunoblotting as described (Zhang et al., 2001). Monoclonal antibodies for GSK-3β, pERK1/2 (T202/ Y204), anti-FLAG M2 and β-actin were obtained from BD Transduction Lab (San Jose, CA, USA), Cell Signaling Technology and Sigma (St. Louis, MO, USA), respectively. Luciferase reporter assay Luciferase reporter assays were performed as previously described (Zhang et al., 2001). For serum starvation/drug treatment, transfected cells were allowed to recover for 16 h before change to serum free medium with kinase inhibitors, as indicated. Luciferase activity was determined by using the Lumat LB9501 luminometer (Berthold Technologies, Oak Ridge, TN, USA). Firefly luciferase activity was normalized to either Renilla luciferase activity or protein concentration. The results were expressed as mean ‘fold induction’. Mean values of at least three independent experiments are displayed±standard deviations.
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Quantitative RT–PCR (qRT–PCR) RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). To produce complementary DNA, 2 μg of total RNA was processed with the Superscript III RT–PCR Kit (Invitrogen), according to the manufacturer’s instructions. Quantitative PCR was performed with the SYBR Green PCR Master Mix using the ABI Prism 7900TM Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). Experiments were performed in triplicate using three independent cDNAs. Primer sequences are provided in Supplementary Table 1. Chromatin immunoprecipitation (ChIP) assays ChIP was performed in Panc 04.03 treated with MAPK inhibitor and BxPC-3 cells transfected with a K-RasG12D, or control expression plasmid using the EZ ChIP kit (Millipore, Billerica, MA, USA). At 16 h post treatment or 36 h post transfection, cells were cross-linked in medium containing 1% formaldehyde for 15 min at 25 °C and harvested in Oncogene. Author manuscript; available in PMC 2012 August 25.
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sodium dodecyl sulfate lysis buffer. Lysed cells were sheared to fragment DNA (200–1000 bp) by sonication (Diagenode, Sparta, NJ, USA). Pre-cleared chromatin was immunoprecipitated with specific antibodies overnight at 4 °C using normal mouse or rabbit IgG as control. ChIP grade rabbit polyclonal antibodies to ETS2 and c-Jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibodies against histone H3 (acetyl K14) and RNA polymerase II were obtained from Millipore. After immunoprecipitation, samples were washed, eluted, cross-links were reversed at 65 °C for 6 h and DNA was isolated and used for PCR. Primer sequences are provided in Supplementary Table 1. Cell proliferation
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Cell proliferation was measured by MTS assay (Promega) and cell cycle analysis. For the MTS assay, 5000–10 000 cells/well were seeded in a 96-well culture plates in quadruplicates and incubated in Dulbecco’s modified Eagle’s medium or RPMI medium containing 10% fetal calf serum supplemented with indicated inhibitors and time periods. Medium was removed and fresh medium was added to each well along with 1:10 dilution of MTS solution. After 2 h incubation, the plates were analyzed with a microplate reader at a wavelength of 490 nm (Molecular Devices, Sunnyvale, CA, USA). Cell cycle analysis was performed using propidium iodide staining (1 mg/ml) and flow cytometry as previously described (Koenig et al., 2010). The DNA content of 50 000 cells was analyzed on a Becton Dickinson FACS Caliber flow cytometer (Beckman Coulter, Brea, CA, USA). The fraction of cells in the G0/G1, S and G2/M phases were calculated using ModFit (Verity Software House, Topsham, ME, USA).
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments Mayo Clinic Pancreatic Cancer SPORE grant CA102701 (DDB and MEF-Z). DDB is a Leukemia and Lymphoma Scholar. AK is supported by a Mildred-Scheel fellowship of German Cancer Society.
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NIH-PA Author Manuscript Figure 1.
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GSK-3β expression is increased in mouse cells overexpressing K-RasG12D. (a) Immunohistochemical staining for GSK-3β of mouse pancreatic tumors expressing KRasG12D under the control of the Pdx-1 promoter. (b) Western blot analysis of GSK-3β expression in mouse pancreatic tumor cells generated by Pdx-1-dependent deletion of BRCA-2−/−/p53−/− genes or expression of K-RasG12D (#1 and #2 represent cell lines from different animals). (c–e) MTS assay of mouse pancreatic cancer cells (c, d) or pancreatic cancer cells obtained from BRCA-2−/−/p53−/− mice and transfected with K-RasG12D or vector control (e) were treated with increasing concentrations of the specific GSK-3 inhibitor LY2064827 for indicated time periods. One representative experiment out of three done in quadruplicates is displayed as mean±s.d., respectively. (d) Sensitivity to treatment with GSK-3i from the experiment in (c) is displayed by normalization of results from cells treated with 2.5 μM LY2064827 to DMSO treated cells, respectively.
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Figure 2.
Mutant K-Ras enhances GSK-3β promoter activity and protein expression via MAPK signaling. (a, d, f) qRT–PCR shows GSK-3β mRNA expression in pancreatic tumor cells obtained from BRCA-2−/−/p53−/− and K-RasG12D expressing mice (a), transfected with two different K-Ras shRNA constructs under serum free conditions for 36 h (d), or treated with 10 μM U0126 for 24 h under serum free conditions (f). All results are displayed as relative to control cells and normalized to RPLP0 expression. (b, c, e) Luciferase reporter assays of indicated pancreatic cancer cells transfected with a −2450/+66 and a −427/+66 GSK-3β promoter constrict (b), cotransfected with the −427/+66 promoter construct and two different K-Ras shRNA constructs for 36 h before analysis (c), or transfected with the −427/+66 construct and treated with 50 μM PD98059, 10 μM U0126, 1 μM wortmannin or 1 μM G06976 for 24 h before analysis (e). Firefly luciferase activity is normalized to Renilla activity. (g, h) Western blot analysis of GSK-3β expression under serum free conditions. ERK-2 or β-actin staining is shown as loading control. HupT3 cells were treated with 100 μM PD98059 or 10 μM U0126 for 48 h (g) or (h) with 10 μM U0126 for indicated time periods (h).
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NIH-PA Author Manuscript Figure 3.
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MAPK signaling is required for Ras-dependent GSK-3β expression. (a–d) Luciferase reporter assay with the −165/+66 reporter construct in BxPC-3 cells cotransfected with pEGFP K-RasG12V, pEGFP H-RasG12V, pEGFP N-RasG12V or vector control, respectively, for 24 h (a); HupT3 cells cotransfected with K-RasG12V or H-RasG12V or vector control for 24 h (b); HupT3 cells cotransfected with K-RasG12D, H-RasG12V, or vector control normalized to RPLP0 expression, 50 μM PD98059 or 10 μM U0126 for 24 h (c); BxPC-3 cells cotransfected with Ras constructs as specified and treated with DMSO or 10 μM U0126 for 24 h (d). Firefly luciferase activity was normalized to total protein amount. Results are displayed as relative luciferase activity or fold activity relative to vector control. (e) qRT– PCR for BxPC-3 cells electroporated with H-RasG12V, K-RasG12V or vector control and treated with DMSO or 10 μM U0126 for 24 h. GSK-3β mRNA expression is normalized to RPLP0. (f) Western blot analysis for GSK-3β expression and ERK phosphorylation in BxPC-3 cells electroporated with H-RasG12V, K-RasG12V or vector control and treated with DMSO or 10 μM U0126 for 24 h under serum free conditions.
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NIH-PA Author Manuscript Figure 4.
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Mapping of Ras responsiveness to proximal 230 bp region of GSK-3β promoter. (a) Diagram of GSK-3β promoter constructs used in luciferase reporter assays. (b) Luciferase reporter assay of HupT3 or BxPC-3 cells cotransfected with indicated promoter constructs and a Renilla reporter construct for 24 h. (c) Luciferase reporter assay in BxPC-3 cells cotransfected with indicated promoter constructs and K-RasG12V or vector control for 36 h. Firefly luciferase activity was normalized to total protein amount. Results are displayed as ×fold activity relative to vector control.
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Figure 5.
Identification of two ETS consensus sites as Ras-response elements. (a) (b) Luciferase reporter assay of HupT3 cells transfected with indicated GSK-3β reporter constructs and treated with 10 μM U0126 for 36 h (a) or BxPC-3 cells cotransfected with indicated promoter constructs and H-RasG12V or vector control for 36 h (b). (c) Cartoon indicating distribution of the putative ETS and AP1 binding sites within the sequence sections A, B and C, relative to the transcription start of proximal promoter region. (d) Reporter assay of BxPC-3 cells cotransfected with promoter constructs containing indicated mutation(s) within the putative ETS binding sites and H-RasG12D or vector control for 36 h. Firefly luciferase results are normalized to total protein amount and displayed as ×fold activity relative to control cells.
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Figure 6.
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ETS proteins are crucial in mediating Ras/MAPK activation of GSK-3β promoter. (a) Western blot analysis for ETS1 and ETS2 expression in pancreatic cancer cell lines with βactin as loading control. (b–e) Luciferase reporter assay with the −165/+66 reporter construct. (b) Panc 04.03 cells were cotransfected with ETS1, ETS1 (T38A), ETS2, ETS2 (T72A) or vector control. (c) HupT3 cells cotransfected with increasing amounts of dn-ETS (+: 100 ng, + +: 250 ng, + + +: 500 ng) or vector control, (d) BxPC-3 cells cotransfected with control vector, K-RasG12V ETS2 or both or (e) with K-RasG12V, dn-ETS, or vector control for 36 h. Results are normalized to total protein amount and displayed as relative luciferase activity (b, c) or ×fold activity relative to control cells (d, e). (f) qPCR shows the amount of DNA precipitated with the ETS2 antibody or normal rabbit IgG after normalizing to the input. ChIP experiments were performed in BxPC-3 cells electroporated with KRasG12V or vector control and maintained for 36 h.
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Figure 7.
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Ras/MAPK signaling is required for ETS2 loading and transactivation of GSK-3β promoter. (a, b) Luciferase reporter assay of MiaPaca2 cells cotransfected with −427/+66 or −165/+66 reporter constructs and (a) ETS2, p300 or vector control or (b) ETS2, control vector and subsequent treatment with 0.5 μM TSA or DMSO for 18 h. Results are displayed as relative luciferase activity normalized to total protein. (c) Luciferase reporter assay of BxPC-3 cells cotransfected with a −165/+66 construct and K-RasG12V, ETS2, or dn-ETS and treated with 0.5 μM TSA, 10 μM U0126 or DMSO for 18 h. Results are displayed as ×fold activity relative to amount of DNA control cells. (d) ChIP experiments in Panc 04.03 cells treated with 10 μM U0126 or DMSO for 24 h. The relative precipitated with the indicated antibodies or from input was accessed by a semi-quantitative PCR. (e) Western blot analysis for GSK-3β protein expression with β-actin as loading control. BxPC-3 cells were electroporated with KRasG12V, ETS2, dn-ETS or vector control and treated with DMSO or 0.5 μM TSA for 24 h under serum free conditions. (f) MTS assay of BxPC-3 cells transfected with vector control, K-RasG12D, or K-RasG12D and Ets-2 were treated with either 2.5 μM LY2064827 or DMSO, for indicated time periods. A representative experiment done in quadruplicates is displayed as mean±s.d.
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