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Epidermal growth factor receptor (EGFR)-targeting therapeutics have shown efficacy in the treatment of colorectal cancer patients. Clinical studies have revealed that activating mutations in the KRAS protooncogene predict resistance to EGFR-targeted therapy. However, the causality between mutant KRAS and resistance to EGFR inhibition has so far not been demonstrated. Here, we show that deletion of the oncogenic KRAS allele from colorectal tumor cells resensitizes those cells to EGFR inhibitors. Resensitization was accompanied by an acquired dependency on the EGFR for maintaining basal extracellular signal-regulated kinase (ERK) activity. Deletion of oncogenic KRAS not only resensitized tumor cells to EGFR inhibition but also promoted EGF-induced NRAS activation, ERK and AKT phosphorylation, and c-FOS transcription. The poor responsiveness of mutant KRAS tumor cells to EGFR inhibition and activation was accompanied by a reduced capacity of these cells to bind and internalize EGF and by a failure to retain EGFR at the plasma membrane. Of 16 human colorectal tumors with activating mutations in KRAS, 15 displayed loss of basolateral EGFR localization. Plasma membrane localization of the EGFR could be restored in vitro by suppressing receptor endocytosis through Rho kinase inhibition. This caused an EGFR-dependent increase in basal and EGF-stimulated ERK phosphorylation but failed to restore tumor cell sensitivity to EGFR inhibition. Our results demonstrate a causal role for oncogenic KRAS in desensitizing tumor cells not only to EGFR inhibitors but also to EGF itself.
The epidermal growth factor receptor (EGFR) is widely expressed in the gastrointestinal tract and stimulates proliferation of a range of cell types, including epithelial cells . Most colorectal tumors are initiated by inactivating mutations in the tumor suppressor gene APC . Loss of functional APC is sufficient to initiate the formation of intestinal polyps in mice, and this is accompanied by increased EGFR expression and activity . Partial loss of EGFR function, or pharmacological inhibition of the EGFR, greatly reduces polyp development in this model . The EGFR is also frequently overexpressed in human colorectal tumors when compared with normal intestinal tissue, and this is associated with increased metastatic potential and poor prognosis [5–7]. EGFR-targeting therapeutics have shown promising clinical activity in a minority of colorectal cancer patients [8–12]. The presence of activating mutations in the KRAS gene in these tumors is a reliable predictor of tumor resistance to anti-EGFR therapy [13,14]. Conversely, high expression of EGFR ligands predicts response to anti-EGFR therapy but only in the subset of wild-type KRAS tumors [15,16]. Although these clinical studies have firmly associated activating mutations in KRAS with resistance to EGFR-targeted therapy, so far, it has not been demonstrated that signaling by the KRAS oncoprotein is the underlying cause of resistance to EGFR inhibition. For instance, it is possible that colorectal tumors with KRAS mutations preferentially develop in an (epi)genetic background of EGFR independence. Such EGFR independence has previously been shown in a minority of tumors that are driven by APC loss only . Constitutive activation of KRAS and its downstream signaling pathways may reduce the dependency on upstream activators such as the EGFR. However, the EGFR activates multiple distinct mitogenic signaling pathways of which the GRB2/SOS/RAS pathway is only one . In addition, activation of the extracellular signal-regulated kinase (ERK) pathway by EGFR ligands is very different in time and amplitude than activation of this pathway by a constitutively active endogenous KRAS mutant protein. For these reasons, we set out to assess the causal relationship between the presence of endogenous oncogenic KRAS and EGFR independence.
The colorectal cancer cell lines HCT116, CT26, and DLD1 were purchased from ATCC (Manassas, VA). The HCT116 cells lacking KRASD13 (HKH2) with their own HCT116 control and the DLD1 cells lacking KRASD13 (DKO4) with their own DLD1 control were obtained from Dr Shirasawa and were previously described . We previously established CT26 cell lines in which the endogenous KrasD12 allele is stably suppressed by mutant-specific RNA interference, using a lentiviral vector (CT26-KrasKD) . Control CT26 cells were transduced with a lentiviral short hairpin RNA (shRNA) construct targeting luciferase (see below). All these cell lines were cultured in Dulbecco's modified Eagle medium (DMEM; Dulbecco, ICN Pharmaceuticals, Zoetermeer, The Netherlands) supplemented with 5% (vol./vol.) fetal calf serum, 2 mM glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin. L145 cells were derived directly from a tumor biopsy of a patient operated on for colorectal liver metastases in our hospital. The tissue fragment was washed with PBS and was mechanically dissociated. Enzymatic digestion (thermolysin [Sigma, St Louis, MO] 0.05% for 2 hours at 37°C) was performed in DMEM/F12. Single-cell suspensions were obtained by filtering through a 40-µm-pore size nylon cell strainer (BD Falcon, Breda, The Netherlands). Spheroids formed spontaneously by culturing in DMEM/F12 (Gibco, Breda, The Netherlands) supplemented with 0.6% glucose (BDH Laboratory Supplies, Soulbury, UK), 2 mM l-glutamine (Biowhittaker, Walkersville, MD), 9.6 µg/ml putrescin (Sigma), 6.3 ng/ml progesterone (Sigma), 5.2 ng/ml sodium selenite (Sigma), 25 µg/ml insulin (Sigma), 100 µg/ml apotransferrin (Sigma), 5 mM HEPES (Gibco), 0.005 µg/ml trace element A (Cellgro, Manassas, VA), 0.01 µg/ml trace element B (Cellgro), 0.01 µg/ml trace element C (Cellgro), 100 µM β-mercaptoethanol (Merck, Schiphol, The Netherlands), 10 ml of antibiotic-antimycotic (Gibco), 4 µg/ml gentamicin (Invitrogen, Molecular Probes, Leiden, TheNetherlands), 0.002% lipid mixture (Sigma), 5 µg/ml glutathione (Roche, Woerden, The Netherlands), and 4 µg/ml heparin (Sigma). The human intestinal epithelial cells (HIECs) were a kind gift from Dr Beaulieu, and these have been described before . All cells were kept at 37°C in a humidified atmosphere containing 5% CO2.
The following antibodies were from Cell Signaling Technology, Inc, Leiden, The Netherlands: rabbit anti-phospho-mitogen-activated protein kinase (MAPK) p44/42 (Thr 202/Tyr 204), rabbit anti-phopsho-Akt (Ser 473), rabbit anti-EGFR (no. 2232; used for HCT116 and HKH2 cells), and rabbit anti.phospho-EGFR antibodies (Tyr 845, 992, 1068, 1045; nos. 2231, 2234, 2235, and 2237). EGFR phosphorylation status was determined by probing the Western blots with a mixture of antibodies 2231, 2234, 2235, and 2237. For EGFR detection in mouse CT26 and CT26-KrasKD cells, we used rat antimouse EGFR (clone 176436; R&D Systems, Minneapolis, MN). Goat anti-Akt1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary peroxidase-conjugated antibodies were from Dako (Glostrup, Denmark).
Gefitinib (Iressa/ZD1839) was kindly provided by AstraZeneca (Macclesfield, United Kingdom). Cetuximab was kindly provided by Merck (Darmstadt, Germany). Erlotinib (Tarceva/OSI1774) was purchased from LC Laboratories (Woburn, MA). The MAPK/ERK kinase (MEK) inhibitor U0126 was from Promega (Madison, WI), the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and the Rho kinase (ROK) inhibitor Y27632 were from Sigma, and the Rac inhibitor (Rac1-Inh) was from Calbiochem (Darmstadt, Germany).
Cells were plated at a density of 5000 cells/well in DMEM containing 5% fetal calf serum in 96-well plates. Cell viability of treated or mock-treated cells was then analyzed for three to six consecutive days by standard 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazoleumbromide (MTT) assays (Roche Diagnostics, Almere, The Netherlands) according to the manufacturer's instructions.
Cells were plated at day 0 at a density of 200,000 cells/well on six-well plates. After an overnight incubation in serum-free medium, the cells were stimulated with the indicated concentrations of EGF and were harvested after the indicated periods of stimulation. Western blot analysis was performed according to standard procedures. Quantity One (Bio-Rad, Hercules, CA) software was used to quantify the signal intensities and ratios between samples loaded on the same gel.
The RAS-binding domain of RAF1 fused to glutathione-S-transferase and coupled to glutathione-sepharose was used as an affinity matrix for activated RAS. The assay and subsequent Western blot analysis with isoform-specific antibodies were performed exactly as described .
Cells were plated at day 0 at a density of 2.106 cells on 10 cm disks. After overnight serum starvation, cells were stimulated with EGF and reverse transcription.polymerase chain reaction was performed. RNA was isolated using the TRIzol method, and complementary DNA was synthesized by Superscript 2 (Invitrogen). The primers used were as follows:
Cells were seeded in a Lab-Tek Chambered no. 1.0 Borosilicate Coverglass System (NalgeNunc International, Rochester, NY) and were mounted on a Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Inc, Sliedrecht, The Netherlands) for live cell imaging under 5% CO2 at 37°C overnight. LysoTracker and Alexa 488-labeled EGF (Invitrogen) were added to the wells, and images were captured every 10 seconds using a Photometrics CoolSNAP HQ charge-coupled device camera (Scientific, Tucson, AZ). Images were processed using MetaMorph software (Universal Imaging, Downingtown, PA).
Cells were grown on glass coverslips and either were stimulated with EGF or were left unstimulated. Cells were fixed by the addition of 3.7% formaldehyde to the culture medium for 10 minutes. Cells were then permeabilized with 0.05% Triton X-100 in 1% PBS/BSA. Coverslips were blocked in PBS with 3%BSA for 1 hour. Anti-EGFR primary antibodies were incubated at room temperature overnight. Secondary antibodies were incubated for 1 hour at room temperature. Coverslips were mounted using Vectashield with 4′,6-diamidino-2-phenylindole (H-1200; Vector Labs, Burlingame, CA). Confocal images were acquired using a Zeiss Meta Axiovert 200M confocal microscope (Carl Zeiss, Microimaging, Inc), with a 40x 1.3 NA objective.
A tissue microarray (TMA) was used to analyze the expression of EGFR in colorectal tumors. The TMA was constructed as previously described in detail . In brief, samples from surgical resections of 55 patients with colorectal cancer with known KRAS mutation status were selected for the TMA. For each sample, three different cylindrical tissue cores were included in theTMA. Immunostaining was performed using standard procedures. After incubating with 3% hydrogen peroxide, antigen retrieval was achieved by boiling in 10 mMcitrate buffer pH 6.0. Sections were blocked with 5% goat serum in TBS and incubated with a rabbit α-EGFR (no. 2232; Cell Signaling Technology, Inc) at 4°C overnight. The HRP-conjugated secondary antibody (DPVM-55HRP; Immunologic, Duiven, the Netherlands) was detected with 3,3′-diaminobenzidine substrate (D4418; Sigma). Slides were counterstained with hematoxylin, were rinsed with water, dehydrated in ethanol, cleared in xylene, and mounted on coverslips. The intensity and localization of EGFR staining and the percentage of positive cells were determined in each separate tissue core by two independent experienced observers blinded to the cores' identities. Staining intensity was scored as follows: 0 = negative, 1 = weak, 2 = moderate, 3 = strong, 4 = very strong. The percentages of positive cells were categorized as follows: less than 1% as 0, 1% to 25% as 1, 25% to 50% as 2, and greater than 50% as 3. The staining coefficient was determined as the product of the staining intensity and the percentage category, so with a maximum score of 12. The EGFR antibody was validated by Western blot analysis using lysates of colorectal cancer cells and by immunohistochemistry on formalin-fixed paraffin-embedded sections of normal human colon and skin tissue. The staining and scoring procedures were independently repeated by a third independent observer.
Differences between the distinct treatment groups were evaluated using the Student's t test. Asterisks indicate statistical significance on the basis of two-tailed analyses of the data sets. Differences with P < .05 were considered statistically significant.
To assess the causal relationship between oncogenic KRAS and EGFR independency, we made use of colorectal cancer cell lines with an activating mutation in KRAS (HCT116, DLD1, and CT26) and their isogenic derivatives lacking oncogenic KRAS (HKH2, DKO4, and CT26-KrasKD) [18,19]. Treatment of HCT116 and HKH2 cells with the anti-EGFR antibody cetuximab or with the small-molecule inhibitors gefitinib or erlotinib had no effect on parental (KRAS-mutant) HCT116 cells but strongly reduced cell proliferation of KRAS-wildtype HKH2 cells (Figure 1A). In addition, cetuximab only marginally affected DLD1 cell proliferation but strongly inhibited DKO4 proliferation (Figure 1B). Likewise, gefitinib had no effect on control CT26 cells, but it suppressed cell proliferation on knockdown of the mutant KRAS allele (Figure 1C). Cetuximab could not be used in this cell system because it does not recognize mouse EGFR. Gefitinib and cetuximab also strongly reduced the proliferation of primary HIECs  expressing wild-type KRAS. In contrast, neither gefitinib nor cetuximab had any effect on the proliferation of freshly isolated colorectal cancer stem cells with an activating mutation in KRAS (L145; KRASD12; Figure 1D). In all these cases, the cells expressing mutant KRAS were significantly more resistant to cell growth inhibition by EGFR-targeting therapeutics than their isogenic KRAS-deleted/suppressed counterparts (Figure W1). Cell death was not observed in any of the previously mentioned experiments. These results suggest that oncogenic KRAS is causally involved in reducing tumor cell dependency on EGFR activity.
The classic RAS/RAF/MEK/ERK pathway is one of the mitogenic signaling pathways that is activated in response to EGFR stimulation. We first assessed the contribution of EGFR activity to basal levels of ERK phosphorylation. Treatment of cells lacking oncogenic KRAS (HKH2, CT26-KrasKD, and HIEC) with either cetuximab or gefitinib caused a strong decrease in the levels of basal ERK phosphorylation, which corresponds with the observed reduction in cell proliferation (Figures 2, A–C, and W1). In contrast, basal ERK phosphorylation was unaffected in tumor cells expressing mutant KRAS (Figure 2, A–C). Thus, EGFR is a major determinant of basal ERK phosphorylation in the absence but not in the presence of oncogenic KRAS.
EGF stimulation of HCT116 and HKH2 cells showed that the immediate early response gene c-FOS was readily induced in HKH2 cells but not in HCT116 cells (Figure 3A). Oncogenic KRAS strongly suppressed EGF-stimulated activation of ERK phosphorylation (Figure 3B). Because NRAS is an efficient activator of the ERK pathway, we examined how oncogenic KRAS affected EGF stimulation of (wild-type) NRAS activity. EGF strongly stimulated NRAS activity in cells expressing wild type or no KRAS but not in the parental cells expressing oncogenic KRAS (Figure 3C). This suggests that the desensitizing effect of oncogenic KRAS on EGF-stimulated ERK pathway activation lies upstream of RAS activation. Indeed, receptor-independent ERK pathway activation at the level of RAF by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate was unaffected, or even more pronounced, in the presence of oncogenic KRAS (Figure 3D). We conclude that oncogenic KRAS strongly reduces the impact of both EGFR inhibition and activation on ERK phosphorylation.
Next, we tested whether oncogenic KRAS selectively suppressed EGF-stimulated ERK pathway activation or whether other pathways were suppressed as well. Tyrosine-phosphorylated (activated) EGFR binds the p85 subunit of PI3K, which results in the activation of AKT. EGF stimulation of KRAS wild-type and mutant cells shows that this pathway is also strongly suppressed by oncogenic KRAS (Figure 3E).
The previously mentioned results suggest that oncogenic KRAS desensitizes cells to EGF at the level of the EGFR. Indeed, basal and EGF stimulated EGFR phosphorylation was strongly impaired in the presence of oncogenic KRAS (Figure 4A). Live cell imaging using Alexa 488-labeled EGF showed that HKH2 cells readily bound and internalized EGF. A large proportion of EGF ended up in the lysosomes as expected (Figure 4B). In contrast, whereas parental HCT116 cells express EGFR (Figure 4A), they bound and internalized far less fluorescent EGF, although some internalization could still be observed (Figure 4B). Therefore, we examined the localization of the EGFR by immunofluorescence analysis. Strikingly, in parental HCT116 cells, most of the EGFR were found in intracellular vesicles (Figure 4C), and their distribution did not change after receptor stimulation (Figure 4D). In HKH2 cells, however, the EGFR predominantly localized to the plasma membrane (Figure 4C) and was internalized after stimulation with EGF (Figure 4, B and D), or after re-expression of KRASG13D (Figure W2). In line with these results, prolonged stimulation with EGF, or with cetuximab, caused downregulation of the EGFR in cells expressing wild-type KRAS but not in cell expressing mutant KRAS (Figure 4E). Also, in the CT26 cell system, we found that suppression of the oncogenic KrasD12 allele restored plasma membrane localization of the EGFR (Figure 5A). Stimulation of CT26 cells did not affect intracellular localization of the EGFR. However, stimulation of CT26-KrasKD cells caused EGFR clustering and internalization, similar to what was observed in HKH2 cells (Figure 5A). In line with these results, CT26-KrasKD cells, but not CT26 cells, efficiently internalized fluorescent EGF (Figure 5B).
Next, we tested whether the relationship between oncogenic KRAS and altered EGFR localization was also observed in human colorectal tumors. To this end, we analyzed EGFR localization in a panel of 55 colorectal carcinomas with known KRAS mutation status using antibody no. 2232 from Cell Signaling Technology (Figures 6 and W3). Of these tumors, 12 showed normal basolateral EGFR staining, 32 showed diffuse staining throughout the tumor cells, and 11 were negative (Figure 6, A and B). In this panel, 16 tumors had an activating mutation in KRAS. Strikingly, only 1 of these tumors displayed normal basolateral staining, 10 displayed diffuse staining, and 5 had lost EGFR staining altogether (Figure 6B). A χ2 test revealed that the unequal distribution of KRAS mutations in tumors with basolateral EGFR staining (1/12) versus tumors with either negative or diffuse staining (15/43) showed a trend toward statistical significance (P = .073). This result suggests that KRAS mutations may be associated with loss of normal basolateral EGFR localization in human colorectal tumors, which could influence the responsiveness of these tumors to EGFR ligands.
Localization and EGF Signaling but Not EGFR Dependency The previously mentioned results suggest that desensitization of HCT116 and CT26 cells to EGF could be due to the loss of EGFR localization from the plasma membrane. We reasoned that inhibition of KRAS signaling could restore EGFR plasma membrane localization. Therefore, we treated HCT116 cells with inhibitors targeting MEK, Rac, PI3K, and ROK and evaluated the response of these cells to EGF.
PI3K or Rac inhibition completely abolished AKT (but not ERK) phosphorylation, but it did not sensitize the tumor cells to EGF. Likewise, MEK inhibition abolished ERK (but not AKT) phosphorylation, but it did not sensitize the tumor cells to EGF. However, inhibition of ROK sensitized cells to EGF-stimulated phosphorylation of ERK1/2 and AKT, albeit not as efficiently as after deletion of mutant KRAS (Figure 7, A and B). ROK inhibition also restored EGF-induced tyrosine phosphorylation of the EGFR (Figure 7C). Furthermore, immunofluorescence analysis showed that the sensitization of HCT116 cells to EGF by ROK inhibition was accompanied by restoration of EGFR localization to the plasma membrane (Figure 7D). Next, we tested whether restoration of EGF signaling in ROK-inhibited cells would be accompanied by a newly acquired dependency on EGFR signaling. To this end, HCT116 cells were treated with cetuximab or gefitinib and the ROK inhibitor Y27632, either alone or in combination. Figure 7E shows that the proliferation of HCT116 cells was not significantly affected by treatment with the drugs, either alone or in combination, although both the ROK and EGFR inhibitors were effective in inducing and suppressing EGFR-dependent ERK phosphorylation, respectively. Next, we tested the effect of ROK inhibition on EGF signaling in CT26 cells. In line with the results previously mentioned, ROK inhibition by Y27632 promoted ERK phosphorylation by EGF (Figure 7F) and restored EGFR plasma membrane localization in CT26 cells (Figure 7G). Also, in this cell system, Y27632 did not restore tumor cell sensitivity to EGFR inhibition by gefitinib (Figure 7H). Taken together, the results suggest that ROK inhibition restores EGFR plasma membrane localization and EGF signaling, but it does not restore tumor cell dependency on EGFR activity.
The association of KRASmutations with resistance to EGFR inhibitors has been demonstrated in a large number of clinical studies, both in patients with colorectal cancer and lung cancer . Our study shows that oncogenic KRAS is causally involved in mediating resistance to EGFR-targeted therapeutics. Constitutive signaling by oncogenic KRAS may reduce the requirement for EGFR activity as an upstream RAS activator. Although we did not observe reduced basal phosphorylation of the ERK or AKT protein kinases after deletion of oncogenic KRAS, maintenance of the activity of these pathways became dependent on EGFR signaling. This lends support to the hypothesis that KRAS renders cells less dependent on the EGFR for maintaining the activity of these critical signaling pathways, albeit at relatively low basal levels.
Oncogenic KRAS desensitized cells not only to EGFR inhibition but also to EGFR activation by altering its intracellular localization. EGFR localization, internalization, and trafficking are controlled by a complex network of signaling molecules [23,24]. After EGF stimulation, the EGFR is downregulated through ubiquitination and lysosomal degradation. Alternatively, internalized EGFR can recycle back to the plasma membrane or can be retained inside the cell. The reduction in cell surface EGFR in KRAS mutant cells limits its availability to EGF and to EGFR-targeting antibodies. We have so far not been able to identify the KRAS effector pathway(s) that cause(s) altered EGFR localization. Although suppression of ROK signaling partially restored EGFR localization to the plasma membrane and EGF responsiveness, the activity of RhoA or ROK was reduced rather than elevated in mutant KRAS cells when compared with wild-type KRAS cells (W.J.v.H. and M.d.B., unpublished observations). This suggests that basal ROK activity is required for EGFR internalization but that mutant KRAS does not stimulate this pathway to accelerate EGFR internalization.
RNA interference (ERK1, ERK2, ERK1 + ERK2, ARAF, BRAF, CRAF, RalA, and RalB) and inhibitor studies (sorafenib, U0126, LY294002, and Rac1) failed to implicate these classic KRAS effector pathways in desensitizing HCT116 and/or CT26 cells to EGF (W.J.v.H., unpublished observations). Possibly, either a combination of effectors or subtle alterations in effector protein activity or localization are required for altering EGFR localization rather than robust changes in expression levels or activity.
From clinical studies, it has become clear that wild-type BRAF is required for response to EGFR therapy in metastatic colorectal cancer . In addition, the Raf/vascular endothelial growth factor receptor inhibitor sorafenib has been used in combination with EGFR inhibitors in the treatment of several solid malignancies . The rationale for this was to simultaneously target the tumor cells (EGFR inhibition) and the vasculature (vascular endothelial growth factor receptor inhibition). However, sorafenib is also a potent RAF kinase inhibitor, and RAF kinases are critical KRAS-activated signal transducers in lung cancer cells . Therefore, it is possible that sorafenib could sensitize colorectal tumor cells to EGFR inhibition by suppressing KRAS/RAF signaling. Our in vitro results do not support such a simple mechanism because neither sorafenib treatment nor RNAi-mediated suppression of RAF kinases could restore tumor cell sensitivity to EGF or EGFR inhibition (W.J.v.H., unpublished observations). This suggests that other RAS effector pathways and/or a combination of effector pathways mediate KRAS-dependent resistance to EGFR inhibition.
Taken together, mutant KRAS causes intracellular retention of the EGFR, which dampens the tumor cell response to EGF. Interestingly, high levels of EGFR ligands predict tumor responsiveness to cetuximab but only in tumors with wild-type KRAS [15,16]. Our results suggest that in mutant KRAS tumors, the EGFR is likely to be a relatively inefficient signal transducer because of its absence from the basolateral membrane. We propose that oncogenic KRAS fixes the activation state of its effector pathways at levels that are relatively low when compared with those achieved after EGF stimulation of KRAS wild-type cells but are high enough to sustain cell proliferation and viability.
EGFR protein levels, as determined by immunohistochemistry, are not associated with the response of colorectal tumors to cetuximab [28,29]. Our results show that a minor population of human colorectal cancer tumors with wild-type KRAS shows proper polarized basal/basolateral localization of the EGFR, similar to what is observed in normal colon tissue. Interestingly, the effect of EGFR activation or inhibition on colorectal cancer cell proliferation was previously shown to be dependent on cell polarity: Only when stimulated or inhibited at the basolateral side does modulation of EGFR activity affect tumor cell proliferation . Possibly, polarized EGFR staining, rather than total protein levels, may identify a subset of wild-type KRAS tumors that respond to EGFR-targeted therapy. This hypothesis should be tested in the tumors of cetuximab-treated cohorts of colorectal cancer patients.
Restoration of proper EGFR localization and signaling in colorectal cancer cells with oncogenic KRAS is possible (by ROK inhibition), but this does not restore tumor cell dependency on EGFR signaling. EGFR unresponsiveness is therefore uncoupled from EGFR independency. The results suggest that the combination of ROK inhibitors with EGFR inhibitors does not seem to be a logical combination strategy to pursue in the clinic at the moment. Identification of (the combination of) KRAS-activated effector pathways that mediate EGFR independency remains a major challenge for future studies.
1This work was supported by grants from the Dutch organization for Medical Scientific Research (ZON-Mw no. 920-03-382 and no. 920-03-534 to W.J.v.H. and M.W.N.), the PON Foundation (to F.J.H.H.), the Association for International Cancer Research (no. 07-552 to M.T.d.B.), and the Dutch Cancer Society (no. 2009-4367 and 2003-3711 to B.L.E. and M.W.N.).