The Effect of Forced Expression of Mutated K-RAS Gene on Gastrointestinal Cancer Cell Lines and the IGF-1R Targeting Therapy
Mutation in K-RAS (K-RAS-MT) plays important roles in both cancer progression and resistance to anti-epidermal growth factor receptor (EGFR) therapy in gastrointestinal tumors. Insulin-like growth factor-1 receptor (IGF-1R) signaling is required for carcinogenicity and progression of many tumors as well. We have previously shown successful therapy for gastrointestinal cancer cell lines bearing a K-RAS mutation using an anti-IGF-1R monoclonal antibody. In this study, we sought to evaluate effects of forced K-RAS-MT expression on gastrointestinal cancer cell lines representing a possible second resistance mechanism for anti-EGFR therapy and IGF-1R-targeted therapy for these transfectants. We made stable transfectants of K-RAS-MT in two gastrointestinal cancer cell lines, colorectal RKO and pancreatic BxPC-3. We assessed the effect of forced expression of K-RAS-MT on proliferation, apoptosis, migration, and invasion in gastrointestinal cancer cells. Then we assessed anti-tumor effects of dominant negative IGF-1R (IGF-1R/dn) and an IGF-1R inhibitor, picropodophyllin, on the K-RAS-MT transfectants. Overexpression of K-RAS-MT in gastrointestinal cancer cell lines led to more aggressive phenotypes, with increased proliferation, decreased apoptosis, and increased motility and invasion. IGF- 1R blockade suppressed cell growth, colony formation, migration, and invasion, and up-regulated chemotherapy-induced apoptosis of gastrointestinal cancer cells, even when K-RAS-MT was over-expressed. IGF-1R blockade inhibited the Akt pathway more than the extracellular signal-regulated kinase (ERK) pathway in the K-RAS-MT transfectants. IGF-1R/dn, moreover, inhibited the growth of murine xenografts expressing K-RAS-MT. Thus, K-RAS-MT might be important for progressive phonotype observed in gastrointestinal cancers. IGF-1R decoy is a candidate molecular therapeutic approach for gastrointestinal cancers even if K-RAS is mutated. © 2016 Wiley Periodicals, Inc.
INTRODUCTION
Gastrointestinal cancers encompass a variety of diseases, many of whose prognoses are poor. Although only colorectal cancer is listed in the top 10 for incidence rates of cancer in the USA, four gastrointestinal cancers, including colorectal, pan- creas, liver and the biliary tract, and esophageal, are in the top 10 for death rates from cancer [1]. In Japan, there are five gastrointestinal cancers, including colorectal, gastric, pancreatic, hepatic, and biliary tract, in the top 10 for death rates in 2013 [2]. Therefore, we must to seek new therapeutic options for these diseases.Signals from a variety of growth factors are required for tumorigenesis and cancer development in human malignancies [3,4]. Recently, advances in molecular cancer research have brought new therapeutic forces from the bench into clinic. One group of new targets is the receptor tyrosine kinases for which tyrosine kinase inhibitors (TKIs) or blocking monoclonal antibodies (mAbs) targeting the receptor or the ligand exist. Epidermal growth factor receptor (EGFR) is an established target and type 1 insulin-like growth factor (IGF) receptor (IGF-1R) could be the next important molecular target [4–6].Binding of the ligands, IGF-1 and IGF-2, to IGF-1R causes receptor autophosphorylation and activates multiple signaling pathways, including the extracellular signal-regulated kinase (ERK or mitogen- activated protein kinase, MAPK) and phosphatidyli- nositide 3-kinase (PI3-K)/Akt-1 pathways [7,8]. These can stimulate tumor progression and cellular differ- entiation [9]. IGF-1R axis is regulated by IGF binding proteins (IGFBPs) and type 2 IGF receptor (IGF- 2R) [10–12]. IGFs stimulate the proliferation and the survival of gastrointestinal cancer cells [10,13–20]. High serum concentration of IGF-1 increases the risk of developing several cancers [11]. Both IGF ligands and receptor are overexpressed in gastrointestinal tumors [16,19,21,22]. IGF-1R is also important for tumor maintenance in addition to carcinogenic- ity [5,23]. Intestinal fibroblast-derived IGF-2 has been shown to stimulate proliferation of intestinal epithelial cells [24]. IGF-2, in conjunction with IGF- 1R, IGF-1, COX-2, and matrilysin (matrix metal- loproteinase-7), seems to play a key role in the early stages of colorectal carcinogenesis [25,26]. IGF-1R signaling is also critical in tumor dissemination through the control of adhesion, migration, and metastasis. Matrilysin can cleave all six IGFBPs and can thus cause activate IGF signals [27,28].
We have previously reported a positive feedback loop between the IGF/IGF-1R axis and matrilysin in the invasive- ness and progression of gastrointestinal cancers [29]. There are several possible approaches to block the IGF-1R axis. Humanized or human mAbs and TKIs for IGF-1R are available and some are in clinical trials now [30–32]. However, a number of phase III clinical trials for anti-IGF-1R mAbs were discontinued, as those have not shown clear benefit of targeting IGF- 1R for solid tumors, including gastrointestinal carci- nomas [6,33]. We have constructed two dominant negative inhibitors for IGF-1R (IGF-1R/dn: IGF-1R/ 482st and IGF-1R/950st), which act by the distinct mechanism of sequestering ligand from endogenous receptors, and are active when expressed in recombi- nant adenovirus vectors for several gastrointestinalmalignancies [16,22,34–36].Gain-of function point mutation in K-RAS is a critical genetic change, as the incidence of K-RAS mutations (K-RAS-MT) is 40–45% in colon cancer and around 90% in pancreatic cancer [37,38]. K-RAS mutations are a negative predictor of efficacy for EGFR-targeted therapies. Cetuximab, which is a mAb for EGFR, is an important drug for patients with colorectal cancer, however, it is not effective for tumors bearing K-RAS-MT [39,40]. Moreover, several colorectal cancers that achieve an initial response to anti-EGFR therapy ultimately acquire resistance to it. One mechanism of the second resistance is emergence of mutation in K-RAS [41,42]. Thus, we need another target to overcome the resistance for anti-EGFR therapy for gastrointestinal cancers with K-RAS-MT.We have reported that the IGF-1R mAb, figitumu- mab (CP-751,871), inhibited signal transduction, proliferation, and survival of six gastrointestinal cancer cell lines [43]. Moreover, in addition to its monotherapy, the combination with chemotherapies was highly effective against these xenograft tumors on mice. The effect of figitumumab was not influ- enced by the mutation status of K-RAS, which suggests that IGF-1R targeting therapy may have therapeutic value in human gastrointestinal carcinomas even in the presence of K-RAS-MT.
In this study, in order to evaluate the effect of mutated K-RAS gene on the phonotype of gastroin- testinal cancer cell lines with wild-type K-RAS, which might represent the second resistance to anti-EGFR therapy, we transfect mutated K-RAS into two cell lines. To assess the effect of IGF-1R blockade, we treated mutated K-RAS transfectants with dominant negative for IGF-1R.Anti-ERK1(K-23) and anti-k-ras(F234) were pur- chased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IGF1Rb(D23H3), anti-phospho-IGF1Rb/ InsRb(19H7), anti-Akt, anti-phospho-Akt(Ser473), anti-phospho-p44/42-MAPK(Thy202/Tyr204), anti- HA-Tag(C29F4), and PathScan Multiplex Western Cocktail-I were from Cell-Signaling Technology (Bev- erly, MA). 5-Fluorouracil (5-FU) and cisplatin (CDDP) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Recombinant human IGF-1 and IGF-2 was purchased from R&D systems (Minneapolis, MN). An IGF-1R inhibitor, picropodophyllin (PPP) [44], was purchased from Calbiochem (San Diego, CA). Specific-pathogen-free female BALB/cAnNCrj-nu mice, 4 weeks old, were purchased from Sankyo Labo Service (Tokyo, Japan). The care and use of mice were according to our university’s guidelines.Recombinant adenovirus expressing IGF-1R/dn (482 amino acids long, ad-IGF-1R/482st) was gener- ated as described previously by homologous recombi- nation [34,35]. An adenovirus expressing the b-galactosidase gene was used as a control (ad- LacZ). The gastrointestinal cancer cells were infected with adenovirus (moi 30).Cell Lines and Generation of Stable TransfectantsAll human gastrointestinal cancer cell lines, colon adenocarcinomas, RKO; pancreatic adenocarcino- mas, BxPC3, were obtained from ATCC (Manassas, VA) in 2000.
Cells were passaged in Eagle’s Minimum Essential Medium and RPMI-1640 with 10% fetal bovine serum. Both cell lines were authenticated regularly, including morphology, growth curve, and analysis mycoplasma detection, and no phenotype changes were observed through the duration of this study.DNA transfection was performed essentially as described previously [34]. Transfection of K-Ras2 G12V(constitutively active) 3xHA-tagged(N-termi- nus) (Missouri S&T cDNA Resource Center, Rolla, MO) and selective plasmid as pcDNA3 (Invitrogen, Carlsbad, CA) of both RKO and BxPC3 cells was performed with LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. After a few weeks of G418 (Clontech/Takara Bio, Ohtsu, Japan) selection, individual colonies were picked and expanded for further analysis. Trans- fectants containing the selection plasmid pcDNA3 alone were used as controls.Western BlottingWhen the cells became 80% confluent, the culture medium was changed to that without serum. After several hours of serum starvation, cells were treated with 20 or 50 ng/ml IGF-1 or 50 ng/ml IGF-2 5– 10 min. Cell lysates were prepared as described previously [34]. Equal aliquots of lysates (100 mg) were separated by 4–12% SDS-PAGE and immuno- blotted onto polyvinylidene Hybond-P membrane (Amersham, Arlington Heights, IL). Analysis was performed using indicated antibodies, and bands visualized by ECL (Amersham).Cells (3 × 103/plate) were seeded onto 60 mmculture plates and incubated for 24 h. The cells were then infected with ad-IGF-1R/482st or ad-LacZ and were incubated for 14 days. After air-drying, cells were fixedwith methanol and stained with Giemsa solution. Colonies containing 50 cells or more were counted.The caspase-3 fluorometric protease assay was performed following the manufacturer’s protocol (BioVision, Milpitas, CA).
In brief, the caspase-3 activity of lysates (100 mg) was measured by colori- metric reaction at 400 nm.Migration AssayWounding assays were performed using a modifi- cation of the procedure described previously [29]. Briefly, six well chambers were prepared by scratching registration marks onto the slide surface. Cells were plated, grown normally for 48 h, and starved over- night. Cells were cut with a cell-scraper and five images were captured 24 h later on an Olympus IX71S1F-2 microscope (Tokyo, Japan) using a 4× objective. For each experiment, two independentobservers counted the number of migrating cells.The assay was performed by a modified Boyden Chamber method as described previously [29]. A total of 1 × 106 cells were seeded on Matrigel-coated filters (8 mm diameter pores, 100 mg/filter; Corning, Tewks- bury, MA). The filters were fixed with cold methanol and stained with hematoxylin and eosin. The cells that had invaded to the lower side of the filters werecounted under a microscope by two independent observers. To assess the effect of IFG-1R blocking, transfectants were infected with ad-IGF-1R/482st or ad-lacZ 2 days before invasion assay.In Vivo Therapeutic Efficacy in Established Tumors on MiceA total of 2 × 105 RKO cells overexpressing mutated K-RAS were subcutaneously injected into nude mice. After tumors became palpable, 2 × 108 PFU of ad-IGF-IR/dn or ad-LacZ were injected intratumorally for 5 successive days. Mice were euthanized when tumors reached 2 cm in size or they developed clinically evident symptoms. Tumor diameters were serially measured with calipers and tumor volume was calculated using the formula: tumor volume (mm3) ¼ (width2 × length)/2.The results are presented as means SD for each sample. The statistical significance of differences was determined by Student’s two tailed t-test in two groups and done by one-way ANOVA in multiple groups, and two-factor factorial ANOVA. P-values of less than 0.05 were considered to indicate statistical significance.
RESULTS
In order to investigate the effect of forced expression of mutated K-RAS on gastrointestinal cancer cell lines, which might reflect the second resistance to anti-EGFR therapy, we transfected HA- tagged K-Ras2 G12V(constitutively active) into two gastrointestinal cancer cell lines, a pancreatic cancer cell, BxPC3 and a colorectal cancer cell line, RKO. K-RAS-MT were stably transfected into the two cell lines and referred to as BxPCrasMT and RKOrasMT, respectively. Specific proteins from the transfected HA-tag were detected by Western blotting in the transfectants (Figure 1a). Controls were empty HA vector transfected and referred to as BxPCcont and RKOcont, respectively.IGF Induced Signal TransductionAfter several hours of serum starvation (from none to 6 h as indicated), both cells were stimulated with 20 ng/ml IGF-1, then extracted proteins were evalu- ated by Western blot assay. IGF-1 induced phosphor- ylation of IGF-1R between 2 and 6 h starvation in were stimulated with 50 ng/ml IGF-1 several minutes from none to 30. The extracted proteins were assessed by Western blotting. pERKs, phosphorylated ERKs; tERKs, total ERKs. (d) All cells (BxPCcont, BxPCrasMT, RKOcont, and RKOrasMT) were cultured with serum (C), without serum (SF), SF then stimulated with 50 ng/ml IGF-1 (1) or 50 ng/ ml IGF-2 (2). The extracted proteins were evaluated by Western blotting. BxPC3 and between 2 and 4 h in RKO (Figure 1b). Thus, we used 4 h starvation in the following experi- ments. IGF-1 also phosphorylated Akt-1. After 4 h starvation, RKO cells were stimulated with 50 ng/ml IGF-1 from none to 30 min before analysis. Both Akt-1 and ERKs were phosphorylated (Figure 1c), within minutes of IGF-1 addition.
IGF-1R was phosphory- lated by 50 ng/ml IGF-1 in BxPCrasMT more than BxPCcont (Figure 1d). Akt-1 was phosphorylated by serum (complete media), 50 ng/ml IGF-1, and 50 ng/ ml IGF-2. Akt-1 was phosphorylated more in K-RAS-MT overexpressing cells than in control cells. Thus, forced expression of K-RAS-MT in both BxPC3 and RKO cells was associated with enhanced IGF signaling, especially of Akt-1. As the ERKs were sometimes phosphorylated by starvation, the effect of mutated K-RAS might be limited compared to Akt.Other Effects of Mutated K-RAS on These Cell LinesThe proliferation of both K-RAS-MT transfectants was higher than those of empty vector transfectants assessed by Trypan blue assay (P ¼ 0.0024 of BxPC3 and P ¼ 0.0238 of RKO, Figure 2a). Colony formation assays revealed that the number of colony of BxPCrasMT was more than that of BxPCcont (P ¼ 0.0004, Figure 2b). The number of colonies of RKOrasMT was also much more than that of RKOcont (P < 0.0001). Scratch assays showed that the motility of BxPC3 cells was increased by forced expression of mutated K-RAS (P ¼ 0.0155, Figure 2c). The accelerating effect of K-RAS-MT on migration was also detected in RKO cells (P ¼ 0.0062). Invasion assays revealed that the number of invading cells was increased by the expression of K-RAS-MT in BxPC3 (P < 0.0001, Figure 2d). Almost the sameresult was observed in RKO (P ¼ 0.0220). Then weassessed the effect of overexpression of mutated K-RAS on chemotherapy-induced apoptosis in RKO. Caspase-3 assay revealed that 500 mM 5FU induced apoptosis in the empty vector transfectant (P < 0.0001, Figure 2e). The 5FU also inducedapoptosis in RKOrasMT cells (P ¼ 0.0014), however,this 5FU-induced apoptosis was suppressed byK-RAS-MT (P < 0.0001 compared to 5FU-treated stimulated invasiveness of both cells. (e) Caspase-3 assay showed that K-RAS-MT reduced 500 mM 5FU-induced apoptosis in RKO cells. K-RAS-MT reduced relative caspase-3 activity of BxPC3 cells without drug. Apoptotic induction by 25 mM Cisplatin (CDDP) tended to be reduced in the presence of K-RAS-MT in BxPC3. cont, control. controls). Relative caspase-3 activity in BxPCrasMT cells was lower than that in BxPC3cont (P ¼ 0.048). Cisplatin-induced apoptosis tended to be down- regulated in K-RAS-MT overexpressing BxPC3.These data indicate that the overexpression of K-RAS-MT gives gastrointestinal cancer cells a more aggressive phenotype. The Effect of IGF-1R Blockade on the Aggressiveness of the Mutated K-RAS Expressing CellsIn order to investigate the effect of IGF-1R blockade, we used ad-IGF-1R/482st and control (ad-LacZ).Colony formation assays revealed that IGF-1R/ 482st effectively reduced the number of colonies of BxPC3cont (P ¼ 0.0045, Figure 3a) and also suppressed K-RAS-MT derived increase in the number of colonies (P < 0.0001). The result was confirmed in RKO cells (P ¼ 0.0012 in empty vector transfectant and P < 0.0001 in K-RAS-MT transfectant, Figure 3b). Scratch assays showed that IGF-1R blockade sup-pressed the number of migrated BxPC3 cells even though K-RAS-MT was overexpressed (P ¼ 0.0049, Figure 3c). K-RAS-MT overdriven mobility of RKO was also reduced by IGF-1R/482st (P ¼ 0.0009, Figure 3d). Boyden chamber assays showed that IGF-1R/dn inhibited the invasiveness of both con- trols, BxPC3 (P ¼ 0.0010, Figure 3e) and RKO(P < 0.0001, Figure 3f). IGF-1R blockade also reducedthe number of invaded cells overexpressing mutated K-RAS in BxPC3 (P < 0.0001, Figure 3e) and in RKO (P < 0.0001, Figure 3f).Then we assessed the effect of IGF-1R blockadeon chemotherapy-induced apoptosis in K-RAS-MT overexpressing BxPC3 cells. Caspase-3 assay revealed that IGF-1R/482st up-regulated 5FU-induced apoptosis (P < 0.0001, Figure 4a), even though BxPC3 expressed mutated K-RAS. IGF-1R/dn also enhanced cisplatin-induced apoptosis in BxPCrasMT (P ¼ 0.0012, Figure 4b).These data suggest that IGF-1R blockade has anti-tumor effects on gastrointestinal cancer cells even if mutated K-RAS gene was overexpressed.The Effect of IGF-1R/dn on Signal Transduction in the Mutated K-RAS TransfectantsWestern blot assays revealed that IGF-1 phosphory- lated IGF-1R and that IGF-1R/482st blocked this phosphorylation in BxPCcont (Figure 5a). In BxPCrasMT, IGF-1R/dn could also block K-RAS-MT upregulated autophosphorylation of IGF-1R. The phos- phorylation of Akt showed almost the same pattern as that of IGF-1R, and IGF-1R/482st inhibited Akt activa- tion in both BxPCcont and BxPCrasMT. However, ERKs were phosphorylated without IGF stimulation in both BxPC3 cells and the effect of IGF-1R/dn was limited. In RKO cells, IGF-1R was autophosphorylated by IGF-1 more in K-RAS-MT overexpressing cells than in controls (Figure 5b). IGF-1R/dn reduced phosphorylation of IGF- 1R, even though K-RAS-MT was overexpressed. IGF-1R/ dn also reduced phosphorylation of Akt-1 in both transfectants.These results suggest that the anti-tumor effects of IGF-1R/482st on mutated K-RAS overexpressing gas- trointestinal cancer cells are mediated by blocking the Akt signal pathway.The Effect of IGF-1R/dn on Xenograft of Mutated K-RASTransfectantsIn order to assess the effect of IGF-1R/482st on mutated K-RAS overexpressing RKO cells in vivo, RKOrasMT cells were inoculated in nude mice and allowed to form evident tumors. Intra-tumoral injec- tion of ad-IGF-1R/482st for 5 successive days markedly suppressed tumor growth (P ¼ 0.031, Figure 6) without influencing body weight.The result suggests that IGF-1R signaling inhibition with this decoy receptor is a candidate new therapeu- tic approach for gastrointestinal tumors with mutated K-RAS. The Effects of Pharmacologic IGF-1R Blockade on the Mutated K-RAS Expressing CellsIn order to investigate the effect of another strategy for IGF-1R blockade, we used an IGF-1R inhibitor, picropodophyllin (PPP) [44]. Colony formation assay showed 60 nM PPP reduced the number of BxPCrasMT colonies but not statistically significantly so (Figure 7a). Colony formation of RKOrasMT was down-regulated by 60 nM PPP (Figure 7b, P ¼ 0.0429). Caspae-3 assay revealed 60 nM PPP enhanced 5FU- induced apoptosis in both mutated K-RAS over- expressing BxPC3 (Figure 7c, P < 0.0001) and RKO (Figure 7d, P ¼ 0.0001). Then, the effect of 60 nM PPPon signal transduction of RKOrasMT was assessed byWestern blotting. PPP reduced both phosphorylation of IGF-1R and Akt-1 but did not that of ERK (Figure 7e).These results suggest that pharmacologic IGF-1R signaling blockade might also be a candidate strategy for gastrointestinal tumors with mutated K-RAS. DISCUSSION Gastrointestinal carcinomas are often diagnosed in advanced stages having lymph node/distant metasta- ses and peritoneal dissemination, and in these cases there are extremely limited options. Gastrointestinal malignancies show a high mortality rates compared to their incidence rates [1]. There are several genetic and epigenetic changes in gastrointestinal tumors relevant to this study, including K-RAS mutation, hypermethylation of IGFBP-3 promoter, and IGF2 differentially methylated region (DMR) 0 hypome- thylation [22,37,45]. Continuous K-RAS activation accelerates cell growth and evokes a feedback system through IGFBP-4/2 to prevent excessive growth in non-transformed lung epithelial cells [46]. However, this growth regulation is disrupted in lung cancer cell lines because of promoter hypermethylation of IGFBP-4/2 genes. The close relationship between ras prosurvival and proapoptotic signaling is coordinated via the differential regulation of the MST2-LATS1 interaction by transient and chronic stimuli in cancer cells [47].In order to assess the oncogenic effect of mutated K-RAS, this gene was transfected or was knocked-in into non-transformed cell usually in the past stud- ies [48]. However, there are limited reports in which mutated K-RAS was transfected into the cancer cell lines [47]. Cancer cell lines usually have a complex accumulation of genetic changes. Although their K-RAS gene is wild type, both RKO and BxPC3 have multiple mutations, such as BRAF, NF1, and PIK3CA in RKO and MAP2K4, SMAD4, and TP53 in BxPC3. In this study, we have transfected mutated K-RAS gene into those cell lines as a model for the emergence of K-RAS-MT in acquired resistance for anti-EGFR thera- pies [41,42]. In the presence of this additional gene product, these cell lines have acquired a more aggressive phonotype. The mutated K-RAS trans- fectants increased proliferation, migration, invasive- ness, and resistance to apoptosis compared to the empty vector transfectants. It might suggest that accumulation of gene mutations such as K-RAS contributes to the progression of tumor. IGF-mediated growth-responsiveness is found in most gastrointestinal cancer cells, including esopha- gus, intestine, pancreas, liver, and biliary tract [13,16,17,19,22]. High expression of both IGF- 1R and the ligands in tumor tissues has indicated continuous activation of this system by paracrine and autocrine loops [19,21]. The expression of IGF-1R/ IGF-2 might be useful as a predictive marker of recurrence and poor prognosis in ESCC [16]. The functional significance of IGF-1R has been demon- strated as cancer growth inhibition is observed with anti-IGF-1R antibodies [19,49]. Furthermore, a new role of the IGF-1R has been identified that there is significant crosstalk between the IGF-1R and other tyrosine kinase receptors. For example, many patients with breast cancer who achieve an initial response to trastuzumab acquire secondary resistance, and one mechanism of resistance has been demonstrated to be overexpression of IGF-1R [50] and another is the formation of IGF-1R/Her2 heterodimers [51].Cetuximab is a key molecular targeted drug for patients with colorectal cancer, however, it shows limited or no efficacy for K-RAS mutated can- cers [39,40]. There is limited information about IGF- 1R-targeted therapy for K-RAS-mutated cancers. In our previous study, figitumumab showed anti-tumor effects for cancers with mutated K-RAS as well as those with wild type both in vitro and in vivo [43]. Our previous data in gastrointestinal carcinomas also indicated that IGF-1R blockade could inhibit Akt signals more than ERK signaling, thus the PI3-K/Akt pathway might play more important role than the ras/ ERK pathway in the downstream signals of the IGF/ IGF-1R axis [16,34–36]. On the other hand, the limited effect on ERK signaling in clinical single agent studies might be one of the reasons why some clinical studies had stopped that the effect of anti-IGF-1R therapies on ERK signals is not strong enough. In this study, we revealed that IGF-1R targeting therapy with a dominant negative receptor has potent anti-tumor effects for gastrointestinal cancer cells overexpressing K-RAS-MT. IGF-1R/482st blocked au- tophosphorylation of IGF-1R and its downstream signal of Akt-1 but only slight inhibition of the ERKs, as shown in our previous studies. Moreover, IGF-1R/ dn enhanced chemotherapy-induced apoptosis in K-RAS-MT transfectants. Resistance to chemotherapy is a serious problem in patients with gastrointestinal malignancies [41,42] and this approach has potential for overcoming this difficulty. It might suggest that targeting IGF-1R signaling is an option to treat patients with gastrointestinal carcinoma, even those that are K-RAS mutated, and that the decoy receptor/ anti-ligand approach might be the most effective strategy.In addition, an IGF-1R inhibitor PPP was found to block IGF/IGF-1R signaling and reduce aggressiveness of K-RAS-MT transfectants, supporting the hypothesis that IGF-1R could be a therapeutic target for gastroin- testinal cancer cells overexpressing K-RAS-MT.One of the limitations of this study is that we used only one K-RAS mutation G12V. Although codon 12 and codon 13 are two major sites of K-RAS gene, many different mutation sites are seen in clinical gastroin- testinal cancers. Different K-RAS mutations might affect detectably different functions. Another limita- tion in this study is that each cancer cell line contains a different genetic background, so those background mutations might also affect the functions observed after transfection of the mutated K-RAS gene. Further studies will be needed to clarify these issues. In the current study, we demonstrate that IGF-1R targeting therapy using transduced decoy receptors suppresses the tumorigenicity and progression of gastrointestinal cancers with K-RAS-MT, by blocking Akt-activation. This study thus validates IGF-1R as a potential therapeutic target in gastrointestinal can- cers and suggest that IGF-1R blockade by ligand sequestration may be a promising anti-tumor therapeutic for these Picropodophyllin diseases.