Promising Therapeutic Efficacy of GC1118, an Anti-EGFR Antibody, against KRAS Mutation-Driven Colorectal Cancer Patient-Derived Xenografts

Epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies, including cetuximab and panitumumab, are used to treat metastatic colorectal cancer (mCRC). However, this treatment is only effective for a small subset of mCRC patients positive for the wild-type KRAS GTPase. GC1118 is a novel, fully humanized anti-EGFR IgG1 antibody that displays potent inhibitory effects on high-affinity EGFR ligand-induced signaling and enhanced antibody-mediated cytotoxicity. In this study, using 51 CRC patient-derived xenografts (PDXs), we showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. In three KRAS-mutant CRCPDXs, GC1118 was more effective than cetuximab, whereas the two agents demonstrated comparable efficacy against three wild-type KRAS PDXs. Persistent phosphatidylinositol-3-kinase (PI3K)/AKT signaling was thought to underlie resistance to GC1118. In support of these findings, a preliminary improved anti-cancer response was observed in a CRC PDX harboring mutated KRAS with intrinsically high AKT activity using GC1118 combined with the dual PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitor BEZ-235, without observed toxicity. Taken together, the superior antitumor efficacy of GC1118 alone or in combination with PI3K/mTOR/AKT inhibitors shows great therapeutic potential for the treatment of KRAS-mutant mCRC with elevated ratios of high- to low-affinity EGFR ligands and PI3K-AKT pathway activation.


Introduction
At initial diagnosis, approximately 20% of colorectal cancer (CRC) patients present with distant dissemination, which is associated with a high mortality rate, highlighting the importance of effective the expression pattern of EGFR ligands ( Figure 1A). Figure 1B and Figure S1 summarize the clinical and genomic baseline characteristics of 51 stratified CRC patients used to establish a PDX series.

Genomic Characterization of CRC PDX Models and Expression Levels of High-and Low-Affinity EGFR Ligands According to KRAS Status
All patients underwent excisional biopsy of a primary CRC (n = 30, 58.8%) or metastatic lesions (n = 21, 41.2%) ( Figure 1B, left panel). Fourteen (27.5%) and 37 CRC patients (72.5%) were diagnosed with localized (stage I-III) and metastatic disease (stage IV), respectively ( Figure 1B, left panel). The primary tumor was in the right colon (cecum to proximal transverse) in 11 cases (21.6%) and the left colon (distal transverse to rectum) in 39 (76.5%) cases. In one case, the location was unknown (n = 1 and 2%) ( Figure 1B, upper right panel). In general, KRAS gene mutations are predominant among RAS family gene alterations in mCRC (85%), and approximately 90% of KRAS mutations occur within codons 12 and 13 [28]. Here, KRAS mutations were observed in 24 (42.1%) cases ( Figure 1B Low-affinity ligands EREG and AREG are predominant in CRC, whereas only a small fraction of high-affinity ligands is expressed [29]. Low expression levels of AREG and EREG associated with KRAS mutations might indicate a tumor that is less dependent on EGFR and is therefore particularly prone to developing resistance to anti-EGFR MoAbs [6,8,10,20,21]. Moreover, the expression levels of AREG and EREG were found to be significantly decreased in mutant-KRAS cases, compared to those in the wild-type cases [30]. Sustained extracellular signal-regulated kinases (ERK) signaling mediated by KRAS mutations was shown to boost secretion of the high-affinity EGFR ligands HB-EGF and TGF-α, which in turn activated EGFR in an autocrine fashion [31]. The total expression level of each EGFR ligand (nM) did not show any significant association with KRAS mutations as evaluated by ELISA (Table S1 and Figure S2). Notably, consistent with previous reports [30,31], we found that KRAS-mutant PDXs tended to show significantly higher fractions of high-affinity EGFR ligands and lower fractions of low-affinity EGFR ligands (Figure 2A,B), in addition to a higher ratio of high-to low-affinity EGFR ligands, than did the KRAS wild-type PDXs ( Figure 2C,D). This indicates that the distribution of high-and low-affinity EGFR ligands depends on the presence of a KRAS mutation.

GC1118 is More Active Than Cetuximab against KRAS-Mutant CRC PDXs
To compare the effectiveness of GC1118 and cetuximab in vivo, 6 CRC PDXs (three KRAS wild-types and three KRAS mutants; all PIK3CA wild-type) were treated with GC1118 for at least 28 days (Figure 3). To compare the effectiveness of GC1118 and cetuximab in vivo, 6 CRC PDXs (three KRAS wildtypes and three KRAS mutants; all PIK3CA wild-type) were treated with GC1118 for at least 28 days ( Figure 3).   Table S3.

Activation of AKT Signaling Confers Resistance to GC1118 Monotherapy in KRAS-Mutant CRC PDX Models
The combined TGII from a panel of CRC PDXs revealed that GC1118 treatment inhibited tumor growth significantly better than cetuximab in KRAS-mutants ( Figure 3 and Table S2); however, complete tumor regression was not observed. In seven CRC PDXs with varying levels of basal EGFR, AKT, and ERK1/2 activation before GC1118 treatment ( Figure 4A), including an additional CRC-024T model (KRAS G12D; high-affinity ligand, 88.8%; low-affinity ligand, 11.2%; high basal AKT activity) with resistance to GC1118 and cetuximab (TGII-GC1118 = 65.6) (Table S2), the efficacy of GC1118 (TGII) showed a significant positive correlation with basal AKT activity only (Pearson's r = 0.82, p = 0.024) ( Figure 4B).  3 . For quantification, images were acquired and signal intensity of each protein band was quantified using the ImageJ software (NIH, Bethesda, MD, USA) and normalized to β-actin. The activities of EGFR, AKT, and ERK1/2 were determined by normalization with their total pairs, namely pEGFR/EGFR, pAKT/AKT, and pERK1/2/ERK1/2, respectively (B) Pearson's correlation analysis was performed to analyze the correlation between EGFR, AKT, and ERK1/2 activities (X-axis) and the tumor growth inhibition index (TGII, Y-axis) in six CRC PDXs.
PI3K activity is the main predictor of mitogen-activated protein kinase kinase (MEK)-inhibitor resistance in KRAS-driven CRC [33,34] and thus, the additional use of a PI3K inhibitor could overcome resistance to MEK inhibition [35]. Although KRAS can directly activate PI3K signaling by binding to the p110-PI3K subunit, there is increasing evidence that PI3K activation, following MEK inhibition, is correlated with RTK activity, providing the foundation for the use of RTK inhibitors in KRAS-mutant CRC [36]. Based on these findings, we performed preliminary in vivo experiments, evaluating the combination of GC1118 and the dual PI3K/mTOR inhibitor BEZ-235 [27], in a relatively GC1118-resistant CRC-024T model (KRASG12D showing high basal AKT activity ( Figure 5). Here, cetuximab was inactive (TGII = 109.4%, p = 0.600), whereas GC1118 (TGII = 65.6%, p = 0.255) or BEZ-235 (TGII = 67.4%, p = 0.103) alone had moderate antitumor effects ( Figure 5A and Table S2). Furthermore, the combination of the two molecules exerted significant inhibitory effects on tumor growth (TGII = 31.6%; p = 0.007; Figure 5A) with no reduction in body weight ( Figure 5B) and without any other signs. We also confirmed significant inhibitory effects on AKT and ERK1/2 activity using IHC ( Figure 5C,D and Table S4) and immunoblotting ( Figure 5E).  3 . For quantification, images were acquired and signal intensity of each protein band was quantified using the ImageJ software (NIH, Bethesda, MD, USA) and normalized to β-actin. The activities of EGFR, AKT, and ERK1/2 were determined by normalization with their total pairs, namely pEGFR/EGFR, pAKT/AKT, and pERK1/2/ERK1/2, respectively (B) Pearson's correlation analysis was performed to analyze the correlation between EGFR, AKT, and ERK1/2 activities (X-axis) and the tumor growth inhibition index (TGII, Y-axis) in six CRC PDXs.
PI3K activity is the main predictor of mitogen-activated protein kinase kinase (MEK)-inhibitor resistance in KRAS-driven CRC [33,34] and thus, the additional use of a PI3K inhibitor could overcome resistance to MEK inhibition [35]. Although KRAS can directly activate PI3K signaling by binding to the p110-PI3K subunit, there is increasing evidence that PI3K activation, following MEK inhibition, is correlated with RTK activity, providing the foundation for the use of RTK inhibitors in KRAS-mutant CRC [36]. Based on these findings, we performed preliminary in vivo experiments, evaluating the combination of GC1118 and the dual PI3K/mTOR inhibitor BEZ-235 [27], in a relatively GC1118-resistant CRC-024T model (KRASG12D showing high basal AKT activity ( Figure 5). Here, cetuximab was inactive (TGII = 109.4%, p = 0.600), whereas GC1118 (TGII = 65.6%, p = 0.255) or BEZ-235 (TGII = 67.4%, p = 0.103) alone had moderate antitumor effects ( Figure 5A and Table S2). Furthermore, the combination of the two molecules exerted significant inhibitory effects on tumor growth (TGII = 31.6%; p = 0.007; Figure 5A) with no reduction in body weight ( Figure 5B) and without any other signs. We also confirmed significant inhibitory effects on AKT and ERK1/2 activity using IHC ( Figure 5C,D and Table S4) and immunoblotting ( Figure 5E).   Table S4. (D) Representative IHC images of AKT and ERK1/2 signaling activities in the CRC-024T PDX model treated with GC1118 and BEZ-235. (E) Analysis of signaling pathways by immunoblotting for AKT and ERK1/2 signaling activities in the CRC-024T PDX model treated with GC1118 and BEZ-235. For quantification, images were acquired and signal intensity of each protein band was quantified using the ImageJ software (NIH, Bethesda, MD, USA) and normalized to β-actin. The activities of EGFR, AKT, and ERK1/2 were determined by normalization with their total pairs, namely phospho-EGFR/EGFR, phospho-AKT/AKT, and phospho-ERK1/2/ERK1/2, respectively. The results in the graph are shown as SEM. The significant difference between vehicle and each treatment group is indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Discussion
As CRCs differ in clinical presentation, molecular heterogeneity, and the involvement of several molecular pathways and molecular changes [5,37], PDXs represent the fastest and most effective approach to uncover active therapeutic agents for CRC [24][25][26]. In contrast to previous studies, we utilized the PDX platform to evaluate the efficacy of GC1118 and its mechanism of action, as the induction and expression of high-affinity EGFR ligands have been reported to be more prevalent in CRC tumor xenografts than in in vitro cultures [8]. GC1118 is a human anti-EGFR IgG1 antibody that differs from existing anti-EGFR MoAbs, such as cetuximab and panitumumab, in its constant region, affinity, mode of action, and efficacy [8,20], exhibiting superior binding affinity (resulting in ADCC) to both the low-and high-affinity variants of FcγRIIIa compared to cetuximab [8,20]. Moreover, the use of Bagg albino (BALB)/c nude mice with intact innate immune systems could allow for the evaluation of GC1118-mediated ADCC through Fc receptors present on immune effector cells such as macrophages, monocytes, and natural killer cells [8,11,38].
A subset of CRCs lacking KRAS pathway mutations and showing "EGFR addiction" is treatable using two EGFR-targeting MoAbs, namely cetuximab and panitumumab [4,9]. When the oncogenic stimulus occurs downstream, such as in tumors with KRAS mutations, resistance to these therapies arises [4,5,7,12,16,39,40]. KRAS mutations in CRC are associated with a more rapid onset and aggressive metastasis, making it clinically more challenging [16,41,42]. Herein, we showed that efficiently blocking high-affinity EGFR ligands with GC1118 induces superior therapeutic benefits in KRAS mutated CRC PDX platform refractory to cetuximab. In addition, the basal up-regulated AKT pathway was correlated with lower efficacy of GC1118, and our preliminary, promising results indicated that GC1118 combined with the PI3K/mTOR/AKT inhibitor BEZ-235 showed improved antitumor effects on KRAS-mutant tumors with intrinsically high AKT activity with favorable safety, encouraging further studies using novel therapeutic combinations to treat clinically-aggressive KRAS-mutant CRC showing elevated ratios of high-to low-affinity EGFR ligands and PI3K/mTOR/AKT signaling ( Figure 6).
Constitutively active MAPK signaling in KRAS-mutated CRC promotes epithelial-mesenchymal transition and cancer stemness, independent of external EGFR stimulation [43,44]. Further, persistent downstream signaling through the RAS axis due to KRAS mutations can activate multiple processes involved in tumor progression and metastasis without the influence of EGFR and other cell surface receptor kinases. Previous studies have reported a significant association between EREG/AREG expression and cetuximab response in KRAS-wild-type patients, but not in KRAS-mutant patients [6,[8][9][10]20,21,45,46]. Therefore, there is an unmet need for novel EGFR-targeting therapies as alternative treatment options. Our results showed that CRC PDXs harboring KRAS mutations expressed remarkably higher levels of high-affinity EGFR ligands than KRAS-wild-type tumors, suggesting that the expression levels of EGFR ligands could be used as biomarkers to predict the therapeutic response to EGFR-targeting strategies. Although EREG and AREG are predominant EGFR ligands expressed in CRC, and only a small fraction of high-affinity ligands is expressed [29], upon downstream activation of the EGFR/RAS/MAPK axis owing to a mutated KRAS effector, the expression of AREG and EREG ligands would be biologically irrelevant in terms of any benefit from cetuximab [8,20,21,45]. The observed superior antitumor potency of GC1118 over cetuximab against CRC PDXs harboring activating KRAS mutations could be due to the strong inhibitory activity of the interaction between EGFR and high-affinity EGFR ligands [8,20,21], providing a rationale for clinical application of the expression pattern of EGFR ligands as a novel biomarker predictive of the response to GC1118 in treating patients with refractory mCRC. Supporting our work, increased secretion of the high-affinity EGFR ligands TGF-α and BTC by some KRAS-mutant clones has been suggested to be a paracrine resistance mechanism to anti-EGFR antibodies in CRC models [47][48][49]. Considering the significant roles of high-affinity EGFR ligands in modulating the tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests potential therapeutic advantages for GC1118 in terms of efficacy and the range of patients for whom it will be beneficial. Genetic and molecular mechanisms determining the ratio of high-affinity/low-affinity EGFR ligands, other than KRAS mutation status, should be elucidated through further comparative analyses of the therapeutic effects of GC1118 on CRC PDXs secreting mainly high-or low-affinity EGFR ligands using a larger panel of heterogenous CRC PDXs. Constitutively active MAPK signaling in KRAS-mutated CRC promotes epithelialmesenchymal transition and cancer stemness, independent of external EGFR stimulation [43,44]. Further, persistent downstream signaling through the RAS axis due to KRAS mutations can activate multiple processes involved in tumor progression and metastasis without the influence of EGFR and other cell surface receptor kinases. Previous studies have reported a significant association between EREG/AREG expression and cetuximab response in KRAS-wild-type patients, but not in KRASmutant patients [6,[8][9][10]20,21,45,46]. Therefore, there is an unmet need for novel EGFR-targeting therapies as alternative treatment options. Our results showed that CRC PDXs harboring KRAS mutations expressed remarkably higher levels of high-affinity EGFR ligands than KRAS-wild-type tumors, suggesting that the expression levels of EGFR ligands could be used as biomarkers to predict Here, importantly, we found that resistance to GC1118 was associated with increased activation of AKT signaling, suggesting that persistent activation of the PI3K/AKT/mTOR signaling axis by high-affinity EGFR ligands could be a potential feedback and resistance mechanism inducing EGFR inhibition. Although we focused on CRC PDX cases harboring only KRAS mutations to validate the potential of combined PI3K/mTOR/AKT and EGFR inhibition in KRAS-mutant CRC cells with high AKT activity due to several mechanisms such as the ratio of high-to low-affinity EGFR ligands, further investigations on CRC PDXs harboring concurrent mutations in both KRAS and the genes activating PI3K/mTOR/AKT pathway (e.g., PIK3CA) are required to strengthen the importance of PI3K/mTOR/AKT pathway in the resistance to GC-1118. Genetic mutations in the PI3K and MAPK pathways are frequently implicated in CRC [10][11][12]. CRC patients with PIK3CA and KRAS mutations are unlikely to respond to the inhibition of the MEK pathway alone or the PI3K pathway alone but will require effective inhibition of both MEK and PI3K/AKT signaling pathways [12,13,16,34,39,[50][51][52][53][54][55]. For example, BEZ-235, in combination with EGFR inhibitors, is more effective for less mTOR inhibitor-sensitive and EGFR inhibitor-resistant CRC cell lines, especially HCT116 (which harbors KRAS and PIK3CA mutations), as shown in a recent study [39]. Previous findings suggest that acquired resistance to anti-EGFR MoAbs biochemically converges on RAS/RAF/MEK/ERK and PI3K/mTOR/AKT pathways, coupled with cross-talk mechanisms between other members of the EGFR family, such as HER2 and HER3, as well as IGF1R [39,[55][56][57][58][59]. Additionally, it is well established that autophagy is associated with resistance to anti-EGFR MoAb therapy because EGFR stimulates multiple downstream signaling pathways that affect autophagy, including the PI3K-AKT-mTOR axis [7,60]. Combination therapy comprising anti-EGFR MoAbs together with autophagy-inducing PI3K/mTOR inhibitors could be used to develop an active therapeutic strategy for mCRC patients by inducing autophagic cell death [61,62].
Activating mutations in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) are present in 15%-20% of CRCs, and the prevalence of PIK3CA exon 9 and/or exon 20 hotspot mutations increases continuously from rectal (10%) to cecal (25%) cancers, supporting the colorectal continuum paradigm [13,14,55,[63][64][65][66][67][68][69][70][71][72][73]. Coexisting PIK3CA and KRAS mutations, which occur in approximately 8%-9% of CRC cases [55,[66][67][68][73][74][75][76][77][78], predict resistance to anti-EGFR therapy, as well as worse prognosis, in CRC [16,39,52,55,66,68,76,[79][80][81][82][83][84][85][86]. Interestingly, mutations in PIK3CA exon 9 (and to a lesser extent exon 20) are associated with features of the traditional serrated pathway (CpG island methylator phenotype-low (CIMP-low)/KRAS mutation) of tumorigenesis [66,68,76,78]. Insight into KRAS-driven CRCs will stimulate new research to find the best approach to treat this aggressive type of cancer, encouraging further evaluations of novel combination strategies including PI3K/mTOR/AKT inhibitors [39,56,87]. Although only one case was tested in the present study, our data highlight the potential of combined PI3K/mTOR and EGFR inhibition for KRAS-mutant CRC cells with relatively high levels of high-affinity EGFR ligands, although further investigation on the therapeutic efficacy, mode of action, and tolerability of this combination based on additional KRAS-mutant PDX models concurrently harboring other genetic alterations (with different genetic backgrounds) is required. In fact, there were three cases with mutations in both KRAS and PIK3CA among our panel (CRC-017T: KRAS G13D, PIK3A Q546K, TP53 R81X and P27R; CRC-021T: KRAS G13D); however, they could not be used for in vivo validation due to the difficulty in obtaining sufficient PDX cells for in vivo combination efficacy test. The verification of the synergy of GC1118 and BEZ-235 in several KRAS-mutant CRC PDX cases less susceptible to GC1118 by high AKT activity is essential to provide clinical reliability and strong support for our hypothesis, highlighting the potential of combined PI3K/mTOR and EGFR inhibition in KRAS-mutant CRC cells with relative high levels of high-affinity EGFR ligands. Our data highlight the potential of combined PI3K/mTOR/AKT and EGFR inhibition in KRAS-mutant CRC cells with relatively high levels of high-affinity EGFR ligands, with a need for further investigations on the therapeutic efficacy, mode of action, and tolerability for optimizing this combination in additional KRAS-mutant PDX models concurrently harboring other genetic alterations. As the low frequency of these double-mutant cases underscores the need for collaborative international efforts to undertake such drug combination studies, optimizing the design of such clinical trials for CRC requires a detailed knowledge of the prevalence of these respective mutant genotypes.
In summary, the superior inhibitory activity of GC1118 on high-affinity EGFR ligands, for which current clinical antibodies show restricted inhibitory activity, reflects the potential therapeutic advantage of this drug for the treatment of cancer in which high-affinity EGFR ligands are implicated in tumor progression, metastasis, and resistance to current cancer therapeutics. Although future work should focus on the development of predictive biomarkers and hypothesis-driven rational combinations, GC1118 might be of therapeutic benefit, alone or in combination with other agents, for KRAS-mutant mCRCs with elevated ratios of high-to low-affinity EGFR ligands and intrinsic PI3K-AKT pathway activation. Further validation based on mouse trials is required based on an expanded CRC PDX panel to overcome the heterogeneity encountered in the clinic and optimize clinical trial designs and further define a patient enrichment strategy.

CRC Patient Clinical Information
All CRC patients provided informed consent for the use of their tissues in this study, in accordance with protocols approved by the Samsung Medical Center (Seoul, Korea) Institutional Review Boards (IRB 2010-04-004). Sequencing analysis (after polymerase chain reaction (PCR) amplification) was performed on 51 patient-derived tissues to confirm the presence of KRAS, BRAF, PIK3CA, and TP53 mutations. Clinical information derived from histological examination and diagnosis based on biopsies from 51 patients with CRC was provided by the Samsung Medical Center. PCRs were carried out in a 20 µL reaction volume containing 100 ng genomic DNA, 10 pmol of each primer, and Maxime PCR premix (iNtRON Biotechnology, Seongnam, Korea). Bidirectional sequencing was performed using a BigDye Terminator v1.1 kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130XL Genetic Analyzer (Applied Biosystems). Sequence analysis was performed using the software package Sequencher 4.10.1 (Gene Codes Corporation, Ann Arbor, MI, USA).

Establishment of CRC PDXs and Analysis of EGFR Ligand Expression
To evaluate autocrine-derived EGFR ligands (and not paracrine ligands produced by stromal cells in the tumor microenvironment), we implanted CRC tumor fragments obtained from 51 patients into the subcutaneous layer of immunodeficient BALB/c nude mice, generating PDXs, as described previously [88]. Animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Samsung Medical Center. Tumors that reached a volume of 1000 mm 3 were considered tumorigenic. Tumor tissues were isolated from subcutaneous CRC PDXs when the tumor volume reached approximately 200 mm 3 . The tumors were homogenized, extracted in 1 mL lysis buffer (Cell Signaling Technology, Danvers, MA, USA), and centrifuged to remove tissue residue. The supernatant components were measured using multiplex ELISA arrays. Human EGF/HB-EGF/TGF-α/BTC/AREG ELISA kits (Ray Biotech, Norcross, GA, USA) and human EREG ELISA kits (USCN Life Science Inc., Houston, TX, USA) were used according to the manufacturers' protocols to quantify the expression level of each EGFR ligand. When tumors reached approximately 200-250 mm 3 , the animals were randomized into groups based on tumor volume to minimize intragroup and intergroup variation (n = 3-7 mice/group). The start of dosing was defined as day 1, and tumor volumes and body weights were measured twice per week for 28-52 days, depending on the growth of each PDX. Tumor volume was calculated as (length × width 2 ) × 0.52 [32]. Relative tumor volume was normalized to the initial tumor volume on day 1. GC1118 or cetuximab was administered at 50 mg/kg (1 mg/mouse) [8,20]. A vehicle was administered intraperitoneally twice per week, and BEZ-235 was administered at 20 mg/kg (0.4 mg/mouse) orally five times per week [89]. TGII values was used for antitumor efficacy [32]. Mice were monitored daily for signs of toxicity. After sacrificing each mouse, tumor tissue was harvested and divided into two parts, one for IHC examination and the other for protein extraction.

IHC
At the indicated post-treatment times, additional tumor-bearing mice were sacrificed and tumors were harvested to generate formalin-fixed paraffin-embedded (FFPE) specimens. FFPE samples were processed according to conventional experimental protocols for IHC analysis. Specimens were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; Gibco), embedded in paraffin, and cut into sections that were blocked and permeabilized with 0.3% triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and 10% horse serum in PBS. Deparaffinization and antigen retrieval were followed by primary antibody staining and hematoxylin counterstaining. Primary antibodies used to label proteins were as follows: anti-phospho-AKT (Ser473; 1:50) and anti-phospho-ERK1/2 (Thr202/Tyr204; 1:100) (Cell Signaling Technology). These were labeled with secondary antibodies, as previously described [90]. To quantify AKT and ERK activity based on IHC, images were captured with an automatic histologic imaging system (TissueFAXS, TissueGnostics GmbH, Vienna, Austria). The expression of anti-phospho-AKT and anti-phospho-ERK1/2 was quantified by HistoQuest Analysis Software using TissueFAXS system (TissueGnostics) after defining regions of interest. Several parameters, such as nuclei size and intensity of staining, were adjusted to achieve optimal cell detection. Cells were plotted to scattergrams according to human-specific marker signals. Cutoff thresholds were determined using signal intensity of the secondary antibody alone as a negative control. Positive cell counts from images of immune-histolabeled sections were measured by two independent observers blinded to the experimental conditions. Mean values for positive cells counted in five locations were evaluated.

Statistics
Results were analyzed for statistical significance using GraphPad Prism V5.04 software and SPSS v.16 (SPSS Inc., Chicago, IL, USA). All data are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Two-tailed t-tests and one-way analysis of covariance were used to assess the differences between two groups of continuous variables, and p values < 0.05 were considered significant. Pearson's correlation coefficients and two-tailed significance were calculated for each case. An unpaired t-test was used to compare TGIIs between different treatments. We used a key to indicate levels of significance as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conflicts of Interest:
The authors have no potential conflicts of interest to disclose.