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The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a major role in oncogenesis. Cetuximab is an EGFR-blocking antibody that is FDA approved for use in patients with metastatic colorectal cancer (mCRC) and head and neck squamous cell carcinoma (HNSCC). Although cetuximab has shown strong clinical benefit for a subset of cancer patients, most become refractory to cetuximab therapy. We reported that cetuximab-resistant NSCLC line NCI-H226 cells have increased steady-state expression and activity of EGFR secondary to altered trafficking/degradation and this increase in EGFR expression and activity lead to hyper-activation of HER3 and down stream signals to survival. We now present data that Src family kinases (SFKs) are highly activated in cetuximab-resistant cells and enhance EGFR activation of HER3 and PI(3)K/Akt. Studies using the Src kinase inhibitor dasatinib decreased HER3 and PI(3)K/Akt activity. In addition, cetuximab-resistant cells were resensitized to cetuximab when treated with dasatinib. These results indicate that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab.
The epidermal growth factor receptor (EGFR) is a member of the HER family of receptor tyrosine kinases and consists of four members: EGFR (ErbB1/HER1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Stimulation of the receptor through ligand binding activates the intrinsic receptor tyrosine kinase and promotes receptor homo- or heterodimerization with HER family members. EGFR activation leads to the downstream stimulation of several signaling cascades, including Ras/Raf/Erk/MAPK and PI(3)K/Akt that influence cell proliferation, angiogenesis, invasion, metastasis and survival.1 Aberrant expression or activity of the EGFR is identified in many human epithelial cancers including colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and brain cancer. Therefore, the EGFR has emerged as one of the most promising molecular targets in oncology.
Targeting EGFR has been intensely pursued in the last decade and has resulted in the FDA approval of five new molecular targeting agents since 2003 in four distinct solid tumors including metastatic colorectal cancer (mCRC), NSCLC, HNSCC and breast cancer. One molecular strategy of EGFR inhibition has been the development of monoclonal antibodies (mAb, cetuximab and panitumumab) directed against the extracellular domain of the EGFR. This results in (1) blockade of endogenous ligand binding to the receptor, (2) inhibition of dimerization with other HER family members and (3) receptor internalization and degradation. A second approach has been the development of small tyrosine kinase inhibitors (TKIs, erlotinib, gefitinib and lapatinib) that are ATP analogues and compete for the ATP binding site in the tyrosine kinase domain (TKD) of the EGFR. Collectively these strategies of EGFR inhibition have resulted in anti-tumor activity in ~10–20% of cancer patients.
Several recent studies have investigated intrinsic and acquired mechanisms of resistance to EGFR TKIs. The identification of catalytic domain EGFR mutations that predict response to EGFR-TKIs in selected lung cancer patients represents a landmark development in the field.2 Mutation in exon 21 of the EGFR TKD, L858R, may predict increased sensitivity to TKIs, whereas the T790M mutation in exon 20 is associated with acquired resistance to TKI therapy.3 These recent findings suggest that patient selection may be critical for successful therapies using EGFR TKIs.4 Although EGFR TKD mutations appear to correlate with response to the TKIs erlotinib and gefitinib, no such correlation exists for cetuximab response.5
Although EGFR is activated through ligand binding and auto-phosphorylation of its cytoplasmic tail, it is well established that Src, or Src family kinases (SFKs), are necessary for full activation of the EGFR.6 Src is the prototype member of a family of non-receptor tyrosine kinases (nRTKs) including Src, Yes, Fyn, Lyn, Lck, Hck Fgr, Blk and Yrk. These cytoplasmic membrane associated nRTKs are transducers of mitogenic signaling emanating from a number of RTKs including fibroblast growth factor receptor (FGFR), platelet derived growth factor (PDFGR), colony-stimulating factor-1 receptor (CSF-1R) and EGFR.7 Investigations into the molecular interactions between SFKs and EGFR have revealed that SFKs can physically associate with activated EGFR.8–10 This results in a conformational change in the SFK and leads to autophophorylation at Y416 and transient activity.11 This activity leads to the phosphorylation of down stream targets12,13 including the EGFR on tyrosine 845 (Y845).14 Y845 is situated within the activation loop of the catalytic domain of the EGFR in a position that is conserved among other RTKs. Autophosphorylation at this conserved site on other RTKs such as the PDGFR, FGFR, insulin receptor (IR) and CSF-1R is necessary for full biological activity of the receptors. Phosphorylation of Y845 on the EGFR results in the receptors ability to enhance EGFR-mediated mitogenesis by binding and phosphorylating the STAT5b transcription.15 The cooperation between Src and EGFR has been well established in breast cancer, where ~70% of breast tumors have Src and EGFR co-overexpressed. More recently, cooperation between SFKs and EGFR has been demonstrated in other tumor types, most notably in HNSCC and NSCLC.16–18
We have previously described the establishment of a series of cetuximab-resistant clones using the NSCLC line NCI-H226.19 The results from our previous work suggest that acquired resistance to cetuximab reflects dysregulation of EGFR internalization/degradation and subsequent EGFR-dependent activation of HER3.20 Here we report that cells with acquired resistance to cetuximab have dramatically increased levels of SFK activity. This increased activity may be due to cooperation with the EGFR and results in signaling to HER3 and PI(3)K/Akt. Treatment of cetuximab-resistant cells with dasatinib, an FDA approved SFK inhibitor, led to decreased HER3 phosphorylation followed by subsequent by loss of PI(3)K/Akt. Furthermore, our data indicates that simultaneous inhibition of SFKs and EGFR markedly augment the suppression of proliferative and survival pathways in NSCLC with acquired resistance to cetuximab. Taken together these data suggest that SFKs and EGFR cooperate in acquired resistance to cetuximab and suggest a strong rationale for clinical strategies that combine both EGFR and SFK inhibitors.
We have previously described the development and characterization of NCI-H226 NSCLC lines with acquired resistance to cetuximab.20 Briefly, the human NSCLC line NCI-H226 (H226) was continuously exposed to increasing concentrations of cetuximab over six months. Following the development of heterogeneous populations of cetuximab-resistant cells, we isolated individual sub-clones of cetuximab-resistant lines. This process resulted in six stable resistant clones for the H226 NSCLC line designated HC1, HC4, HC5, HC6, HC7 and HC8. The sensitive parental line was designated HP. Three of these clones (HC1, HC4 and HC8) were tested for their sensitivity to increasing concentrations of cetuximab relative to the parental controls (Fig. 1A). All HC clones displayed a robust cetuximab-resistant phenotype when challenged with increasing concentrations of cetuximab as compared to parental controls. Flow cytometric analysis of cell cycle phase distribution showed that cetuximab treatment induced a strong G1 arrest in the parental cells whereas cetuximab had no effect on cell cycle distribution of the HC1, HC4 and HC8 cetuximab-resistant clones (Fig. 1B).
Findings from our previous work indicated that steady state expression and activity of the EGFR was increased in all cetuximab-resistant clones (HC1, HC4 and HC8) relative to the parental control (HP) and is shown in Figure 1C. In addition to increased expression and activity of the EGFR, phospho-analysis of individual tyrosine residues indicated that the tyrosine 1173 (Y1173), an auto-phosphorylation site, and 845 (Y845), a site phosphorylated solely by Src or its family members,6 showed a robust phosphorylation in cetuximab-resistant clones as compared to parental controls (Fig. 1C). This finding led to investigations of SFK activity in several cetuximab-resistant clones. Tyrosine 416 (Y419 in human Src) is an autophosphorylation site that is highly conserved among Src family members.21,22 Phosphorylation at this site results in a conformational change in the kinase activation loop, allowing for substrate binding and full activation of the enzyme. Immunoblot analysis indicated that the active form of SFKs was dramatically increased in all resistant clones relative to the parental line. In addition, cetuximab-resistant clones showed multiple banding patterns using a pan-SFK antibody, indicating the expression of multiple SFK family proteins in resistant cells (Fig. 1C). Furthermore, SFKs kinase assays indicated that cetuximab-resistant cells (clone HC4) had an approximately 2-fold increase in overall SFK activity (Fig. 1D). Taken together these results suggest that cells with acquired resistance to cetuximab have increased SFK activity.
It has been well described that SFKs become active by binding phosphorylated RTKs, including the EGFR, FGFR, PDGFR and the receptor for CSF-1, via their SH2 domains.23 This results in a conformational change leading to the autophosphorylation of Y416 on SFKs and full activation of the kinase. To determine if the elevated steady state expression and activity of the EGFR in cetuximab-resistant cells played a role in the activation of SFKs we treated cetuximab-resistant cells (HC4) with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib or siRNA directed against the open reading frame of the EGFR. Treatment with gefitinib or erlotinib (1 uM) led to decreased phosphorylation of the EGFR as measured by Y1173 and Y845 (Fig. 2A). This treatment resulted in downregulation of the active form of SFKs relative to the control and cetuximab treated groups. Treatment of cetuximab-resistant cells with siRNAs against the EGFR led to the loss of expression of the EGFR as indicated by Western analysis and resulted in significant loss of active SFKs (as measured by Y416), but not the total level of SFKs (Fig. 2A). EGFR knockdown in cetuximab-resistant cells resulted in decreased proliferative potential.19 To build upon these findings and confirm that loss of EGFR results in the loss of SFK activity in cells with acquired resistance to cetuximab we performed SFK kinase assays (Fig. 2B). Cetuximab-resistant cells (HC4) treated with DMSO vehicle control, erlotinib or siRNA directed against the EGFR for 24 hours resulted in decreased SFK activity. Taken together these results indicate that the EGFR is responsible for the activation of SFK in cells with acquired resistance to cetuximab and that these molecules cooperate in the resistant phenotype.
Given the increased amount of SFK activity in cetuximab-resistant cells, we hypothesized that increased SFK activity leads to increased phosphorylation and therefore activity of the EGFR. To test this hypothesis we treated HP parental and HC1, HC4 and HC8 cetuximab-resistant clone with either vehicle control or 50 nM of dasatinib for 24 hours. The EGFR was immunoprecipitated and immunoblotted using an anti-phospho-tyrosine (pTyr) antibody. The three resistant clones exhibited a decrease in total phosphorylation of the EGFR when treated with dasatinib, whereas the parental control showed no change (Fig. 3A). Proliferation analysis using HP, HC1, HC4 and HC8 clones indicated that cetuximab-resistant cells were more sensitive to dasatinib as compared to HP parental cells (Fig. 3B). This decrease in proliferation correlated with decreased SFK activity in the HC4 resistant clone, (Fig. 3C). Dasatinib had very little effect on SFK activity in the HP parental cells, whereas dasatinib treatment of the HC4 cells led to a dramatic decrease in SFK activity restoring it to near parental levels. Taken together these results suggest that cetuximab-resistant cells are highly dependent on SFK activity for proliferation and survival.
In our first report of this model of acquired resistance we showed that EGFR and HER2 activation of HER3 is a critical mechanism for escaping cetuximab therapy. Furthermore, we showed that blockade of the EGFR (using TKIs or siRNAs) lead to decreased HER3 phosphorylation and ultimately the PI3K/Akt survival pathway.20 To determine if SFKs play a role in enhancing signals to HER3, we blocked SFK activity in parental and cetuximab-resistant clones (HP, HC1, HC4 and HC8) and measured its effects on HER3 and PI(3)K/Akt signaling (Fig. 4). Dasatinib treatment resulted in the inactivation of SFK (as measured by Y416 phosphorylation) and subsequent phosphorylation of Y845 of the EGFR (data not shown). These results correlated with reduced HER3 phosphorylation and subsequent PI(3)K/Akt activation in two of three cetuximab-resistant clones (HC1 and HC4). Total levels of HER3 and PI(3)K/Akt were not effected by drug treatment (Fig. 4). Also, treatment with dasatinib led to increased steady state expression of total SFKs. These results indicate dasatinib may be able to abrogate signaling to HER3 and PI(3)K/Akt and ultimately overcome cetuximab resistance.
Next we examined if low doses of dasatinib could re-sensitize cetuximab-resistant cells to cetuximab therapy. We performed proliferation analysis assays using DMSO control, 100 nM cetuximab, 25 nM dasatinib or the combination on HP, HC1, HC4 and HC8. The results of these experiments showed that dasatinib induced mild growth inhibition on cetuximab-resistant cells but the combination of the two drugs showed augmentation of growth inhibition (Fig. 5A). Combination of cetuximab with dasatinib resulted in decreased pHER3 and pAkt (Fig. 5B). These results indicate that dual targeting of the EGFR and its cooperating intracellular kinases, SFKs, might produce greater clinical impact than either agent alone.
Acquired resistance to cetuximab can occur by several distinct mechanisms including (1) mutations in the EGFR that alter response to cetuximab, (2) loss of target (i.e., EGFR), (3) loss of downstream signaling molecules, (4) increase in HER family member ligands, (5) and activation of other RTKs with overlapping signal transduction cascades with EGFR (i.e., cMET, IGF1R). One approach to identifying molecular changes leading to acquired resistance to cetuximab is to develop resistant cells lines by chronically challenging them with cetuximab and evaluating the resulting clones with congenic sensitive parental lines. Previously we reported the development of a cetuximab acquired resistance model using the NSCLC NCI-H226.20 Lung lines with acquired resistance to cetuximab exhibited increased steady-state EGFR expression and activity secondary to alterations in trafficking and degradation.20 This increased steady-state activity resulted in constitutive activation of HER3 and signals to PI(3)K/Akt leading to escape from cetuximab therapy. In the current study, we found that cells with acquired resistance to cetuximab had a higher basal level of active SFKs. This increased SFK activity led to prolonged EGFR activity and was critical for EGFR activation of HER3 and subsequent activation of PI(3)K/Akt and other survival signals. Furthermore, combinatorial treatment of cetuximab-resistant cells with dasatinib could re-sensitize resistant cells to cetuximab growth inhibition. Collectively, these data suggest that SFKs and EGFR cooperate in acquired resistance to cetuximab. Combinatorial therapy targeting these two kinases may have increased clinical efficacy than targeting either molecule alone.
The EGFR plays a crucial role in human malignancies and contributes to tumor formation and metastasis. However, it is well established that Src and its family members can potentiate the transformation ability as well as the mitogenic and tumorigenic activity of the EGFR.6,22,24,25 Full kinase activity of SFKs is a prerequisite for biological cooperation with EGFR as well as for phosphorylation of Y845 on the EGFR. SFK phosphorylation of Y845 on the EGFR is required for DNA synthesis stimulated by EGF as well as endothelin, lysophosphatidic acid, growth hormones and cytokines. Experiments using an EGFR Y845F mutant in mouse fibroblasts resulted in blockade of DNA synthesis.6,15,26 Taken together these findings suggest that Src and its family members are critical for full activation of the EGFR. In addition to direct phosphorylation and cooperation with the EGFR, previous work has shown that SFKs mediate EGFR ligand cleavage leading to proliferation and invasion of HNSCC cancer cells.27 Recent work by Koppikar et al. indicated that combined inhibition of the SFKs and the EGFR abrogated both growth and invasion of HNSCC.18 Collectively these reports provide a persuasive body of evidence indicating a strong relationship between SFKs and the EGFR in cancer.
We found cells with acquired resistance to cetuximab exhibit increased steady-state activity of the EGFR (Fig. 1C and reviewed in ref. 20). This is in contrast to a recent report suggesting increased ubiquitination (and thus decreased EGFR levels) as a mechanism of acquired resistance to cetuximab therapy in DiFi colorectal cetuximab-resistant cells.28 However, similar to their findings we show that cetuximab-resistant cells have increased basal level of active SFKs resulting in hyper-phosphorylation of the Y845 and Y1173 of the EGFR (Fig. 1C). Taken together these results indicate that SFK activation in both NSCLC and CRC cancer lines with acquired resistance to cetuximab may have a common mechanism of resistance.
SFKs become active upon binding to several RTKs including the EGFR, PDGFR, FGFR, CSF-1R, as well as integrins, and cell-cell adhesion molecules.22 Data in Figure 2 indicates that blockade of EGFR through TKIs or siRNA results in the loss of active, but not total, SFK in cells with acquired resistance to cetuximab. Collectively, these data suggest that the EGFR, rather than an alternative RTK, is responsible for the binding and activation of SFKs. These data strengthen the conclusions of this communication that co-targeting SFKs and the EGFR may have increased effectiveness than targeting either molecule alone.
One consequence of increased SFK activity is enhanced EGFR phosphorylation (Fig. 3A), which could be abrogated by treatment with dasatinib in cetuximab-resistant clones, but not parental cells. Not only did this treatment lead to decreased phosphorylation of the EGFR, but also resulted in decreased survival of cetuximab-resistant clones (Fig. 3B). Our data indicates that dasatinib influenced the HER3-PI(3)K/Akt survival pathway in cells with acquired resistance to cetuximab (Figs. 4 and and55).
Currently there are nine known family members of the Src family. When measuring active levels of SFKs in cells with acquired resistance to cetuximab, we observed a different banding pattern when compared to parental controls (Figs. 1C and and4).4). The parental cell lines, when immunoblotted with the SFKY416 antibody, showed a single active band. However, three independent cetuximab-resistant clones had multiple banding patterns. This may suggest that cetuximab-resistant cells have increased activity of several different members of the Src family. This may account for the increased SFK activity in resistant cells. However, dasatinib treatment resulted in loss of the active band(s) in the HP, HC1, HC4 and HC8 clones (Fig. 4) but did not alter proliferative potential of cetuximab sensitive parental lines (Fig. 3B). This suggests that cells with acquired resistance to cetuximab may have acquired multiple active forms of SFKs but also acquired an oncogene addiction to these signaling molecules.
It has been reported that increased SFK activity in NSCLC can promote the survival of EGFR-dependent cell lines by enhancing phosphorylation of the EGFR and HER3.16 We previously reported that escape from cetuximab therapy is centered around activation and recruitment of HER3.20 This escape mechanism has been reported by other investigators.29,30 Blockade of SFK activity by dasatinib, at high doses, in resistant cells lead to decreased phosphorylation of EGFR at Y845 (data not shown) and led to decreased HER3 and subsequent PI(3)K/Akt activity (Fig. 4). These findings support the conclusions by Zhang et al. which reported experiments blocking SFKs in EGFR-dependent NSCLC that resulted in decreased phosphorylation of HER3.16 Furthermore, they concluded that SFKs are active in NSCLC and cooperate with the EGFR to promote survival. Here we demonstrate that SFKs and the EGFR cooperate in acquired resistance to cetuximab. Experiments aimed at modulating SFKs and EGFR levels in cetuximab-sensitive lines are ongoing. Taken together these results indicate that SFKs play a strong role in mediating signals from the EGFR to HER3 and subsequently the PI3(K)/Akt pathway and suggest that increased SFK activity may lead to escape from cetuximab therapy.
In Figure 5 we targeted both the EGFR and SFKs using dasatinib and cetuximab to determine if combinatorial therapy directed at both SFKs and the EGFR would result in increased anti-proliferative activity than either agent alone. Our data suggested that the combination had a profound impact on proliferative potential and that the combination could abrogate active HER3 and PI(3)K/Akt pathways. These data, in conjunction with Lu et al.28 strengthen the hypothesis that SFKs and the EGFR cooperate in both NSCLC and CRC and may be a common molecular mechanism to escape cetuximab challenge.
In conclusion, NSCLC with acquired resistance to cetuximab show a remarkable increase in the activity of Src family kinases. This enhanced activity of SFKs leads to increased activity of the EGFR leading to activation of HER3 and the PI(3)K/Akt and ultimately escape from cetuximab therapy. Treatment of cetuximab-resistant cells with dasatinib results in re-sensitization to cetuximab therapy. Collectively, our data suggest a strong rationale for clinical strategies that investigate combinatorial therapy directed at both the EGFR and SFKs in patients with acquired resistance to cetuximab.
The human NSCLC line NCI-H226 was obtained from ATCC (Manassas, VA). The cells were maintained in 10% fetal bovine serum in RPMI with 1% penicillin/streptomycin. Cell culture media and supplements were obtained from Life Technologies, Inc., (Gaithersburg, MD).
Gefitinib (ZD1839, Iressa™) was generously provided by AstraZeneca (Macclesfield, UK). Erlotinib (OSI-774, Tarceva™) was generously provided by Genentech, Inc., (San Francisco, CA) and OSI. Cetuximab (C225, Erbitux™) was generously provided by ImClone Systems Inc., (New York, NY). Dasatinib (BMS-354825, Sprycel™) was generously provided by Bristol-Myers Squibb (New York, NY).
All antibodies were purchased from commercial sources as indicated below: EGFR, p-EGFR(Tyr1173), and HRP-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were obtained from Santa Cruz Biotechnology Inc., (Santa Cruz, CA). Total SFK, pSFK(Y416), pEGFR(Y845), pHER3(Y1289) and pAKT(S473) were obtained from Cell Signaling Technology (Beverly, MA). Alpha-tubulin was purchased from Calbiochem (San Diego, CA) and pTyr (4G10) from Millipore (Billerica, MA).
Previously described in reference.20 Briefly, H226 tumor cells were continuously exposed to increasing concentrations of cetuximab. Commencing with the IC50 of cetuximab, the exposure dose was progressively doubled every 10–14 days until 8 dose doublings had been successfully achieved. In parallel, controlled parental cells were exposed to the PBS vehicle. The established resistant cell lines were then maintained in continuous culture with the maximally achieved dose of cetuximab.
Exponentially growing cells were seeded in 6 well plates. Following treatment, monolayers were washed with PBS and fixed/stained with 0.5% crystal violet. Plates were air dried overnight and dye was eluted with 0.1 M sodium citrate (pH 4.2) in ethanol (1:1). Elution was transferred to 96-well plates, and the absorbance was read at 540 nm to determine cell growth. The anti-proliferative effect of dasatinib (Fig. 3) was evaluated using MTT assays. Briefly, exponentially growing cells were seeded into 96-well plates and incubated in medium containing various concentrations of dasatinib for 24 hours at 37°C. Duplicate plates containing six replicate wells/assay were seeded at a density of 1,500 cells in 0.1 ml of medium. Dasatinib was solubilized in DMSO. Final concentration of DMSO in all cultures, including controls, was 0.1%. After exposure of cells to dasatinib, 100 μl of MTT (1 mg/ml) was added to each well for 2 hours at 37°C to allow MTT to form formazan crystals by reacting with metabolically active cells. The formazan crystals were solubilized overnight at 37°C in a solution containing 10% of SDS and 50% of N,N-dimethylformamide. The absorbance of each well was measured in a microplate reader at 600 nm. The percentage cell growth was calculated by comparison of the A600 reading from treated versus control cells.
Following treatment, cells were lysed with Tween-20 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml of leupeptin and aprotinin), sonicated and protein was quantitated using a standard Bradford absorbance assay. Equal amounts of protein were fractionated by SDS-PAGE. Thereafter, proteins were transferred to a PVDF membrane and analyzed by incubation with the appropriate primary antibody. Proteins were detected via incubation with HRP-conjugated secondary antibodies and ECL chemiluminescence detection system.
Cells were lysed with NP-40 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Deoxycholic acid, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml of leupeptin and aprotinin). Cell lysates containing 0.5 mg of protein were incubated overnight at 4°C with 2 μg of anti-EGFR antibody. After adding 30 μl of protein A/G agarose beads, cell lysates were incubated for another 2 hours at 4°C. The immunoprecipitates were pelleted by centrifugation and washed three times with NP-40 buffer. The captured immunocomplexes were then eluted by boiling the beads in 2× SDS sample buffer for 5 minutes and subjected to immunoblot analysis as described above.
For cell cycle distribution analysis, cells were plated at a density of 500,000 per 100 mm plate and allowed to attach overnight. The cells were treated with either PBS vehicle or 100 nM cetuximab for 24 hours. The following day the cells were pulsed with 10 uM BrdU for one hour. The cells were harvested by trypsinization, washed with cold PBS and fixed with 70% ethanol for 20 minutes. The cells were then labeled with a FITC-conjugated mouse anti-BrdU antibody and processed according to manufacturer recommendation (BD Pharmingen, San Jose CA). The cells were analyzed by flow cytometry (BD FACScan). ModFit Software (Verity Software House, Topsham, ME) was used to analyze the data.
To measure the activity of Src family kinases the Omnia™ Tyr recombinant kit was used (Biosource, Camarillo, CA). Cetuximab-sensitive and resistant cells were grown to 70% confluence and lysed in in NP-40 lysis buffer buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% Deoxycholic acid 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml of leupeptin and aprotinin]. Cell lysates containing 1 mg of protein were incubated overnight with 2 μg of pan-anti-Src antibody (IP-activity Src Kit, Calbiochem, San Diego, CA). Lysates were then incubated with 30 μl of proteinA/G agarose beads for 2 hours. The immunoprecipitates were pelleted by centrifugation and washed three times with 1× kinase buffer. The kinase reactions were performed according to manufacturer instruction. Briefly, the kinase activity of SFKs is measured by the phosphorylation of a novel peptide substrate that contains a chelation-enhanced fluorophore. Upon phosphorylation the substrate is chelated resulting in an increase in fluorescence. Fluorescent intensity readings were read in triplicate after 30–60 minutes.
All siRNAs were obtained from Dharmacon (Lafayette, CO.,). Transfection of Dharmacon siRNA into cell lines was achieved by using Dharmafect 1 reagent from Dharmacon.
We would like to thank AstraZeneca (gefitinib), BMS (dasatinib), ImClone (cetuximab) and OSI/Genentech (erlotinib), for supplying EGFR and SFK targeting agents. This work was supported in part by NIH R01 CA 113448-01 (P.M.H.) and American Cancer Society postdoctoral fellowship PF-07-089-01-TBE (D.L.W.).
P.M.H. holds research and/or consulting agreements with Amgen, AstraZeneca, Cellectar, Genentech and ImClone.