Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer
© Min et al.; licensee BioMed Central. 2015
Received: 18 December 2014
Accepted: 21 May 2015
Published: 4 June 2015
Therapeutic interventions in the insulin-like growth factor receptor (IGF-1R) pathway were expected to provide clinical benefits; however, IGF-1R tyrosine kinase inhibitors (TKIs) have shown limited antitumor efficacy, and the mechanisms conveying resistance to these agents remain elusive.
The expression and activation of the IGF-1R and Src were assessed via the analysis of a publicly available dataset, as well as immunohistochemistry, Western blotting, RT-PCR, and in vitro kinase assays. The efficacy of IGF-1R TKIs alone or in combination with Src inhibitors was analyzed using MTT assays, colony formation assays, flow cytometric analysis, and xenograft tumor models.
The co-activation of IGF-1R and Src was observed in multiple human NSCLC cell lines as well as in a tissue microarray (n = 353). The IGF-1R and Src proteins mutually phosphorylate on their autophosphorylation sites. In high-pSrc-expressing NSCLC cells, linsitinib treatment initially inactivated the IGF-1R pathway but led a Src-dependent reactivation of downstream effectors. In low-pSrc-expressing NSCLC cells, linsitinib treatment decreased the turnover of the IGF-1R and Src proteins, ultimately amplifying the reciprocal co-activation of IGF-1R and Src. Co-targeting IGF-1R and Src significantly suppressed the proliferation and tumor growth of both high-pSrc-expressing and low-pSrc-expressing NSCLC cells in vitro and in vivo and the growth of patient-derived tissues in vivo.
Reciprocal activation between Src and IGF-1R occurs in NSCLC. Src causes IGF-1R TKI resistance by acting as a key downstream modulator of the cross-talk between multiple membrane receptors. Targeting Src is a clinically applicable strategy to overcome resistance to IGF-1R TKIs.
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide , and the 5-year survival rate for patients with advanced NSCLC remains less than 20 % . Although a small subset of patients with specific genetic and epigenetic abnormalities has shown clinical response to specific targeted therapies , most patients with NSCLCs are insensitive to chemotherapy and radiation1. Moreover, acquired resistance to the therapies eventually emerges in the initially sensitive patients after continuous treatment . As activation of the survival potential seem to provide cancer cells resistance to anticancer therapies, it is likely that effective anticancer therapies must occur in parallel with blockade of key survival pathways.
The insulin-like growth factor receptor (IGF-1R) signaling pathway plays a critical role in cancer cell survival, causing resistance to numerous anticancer drugs [5–7]. Therefore, effective regimens to inactivate the IGF-1R pathway may sensitize cancer cells to anticancer therapies and provide clinical benefits to cancer patients. However, patients who show a promising initial response to anti-IGF-1R monoclonal antibodies (mAbs) appear to rapidly acquire resistance to these mAbs [8–10]. Similarly, therapeutic efficacy of IGF-1R TKIs has been modest in a variety of human cancers, including NSCLC2 thus, there is an urgent need to understand the signaling pathways that confer inherent and/or acquired resistance to anti-IGF-1R drugs and to develop new strategies to overcome this resistance.
The canonical activation of IGF-1R results from the binding of ligands (IGF1 and IGF2) to IGF-IR, insulin receptor (IR) (with a lower affinity than insulin), and hybrid receptors of IGF-1R/IR [6, 11], leading to the autophosphorylation of tyrosine residues 1131, 1135, and 1136 in the activation loop of the IGF-1R β-chain (the corresponding residues in the human IR are 1158, 1162, and 1163, respectively). Previous reports have suggested that Src, a non-receptor protein tyrosine kinase, can directly phosphorylate IGF-1R [12, 13]. Src also plays an important role in cancer cell survival and resistance to targeted anticancer therapies by acting as a common signaling facilitator that is activated by a myriad of redundant signaling pathways [12, 14]. The elevated expression and/or activation of Src and multiple membrane-associated receptors, such as IGF-1R, IR integrins, HER2/neu, and EGFR (all of which trigger Src activation), have been reported in NSCLCs [14–17]. Hence, it is likely that Src acts as a downstream node that links signalings among several collateral membrane associated receptors.
Here, we demonstrate high levels of Src and IGF-1R co-activation though mutual phosphorylation in the majority of NSCLC. We show that Src kinase activity plays a key role in de novo resistance to IGF-1R TKIs in NSCLC cells. NSCLC cells with high Src kinase activity can be independent from IGF-1R activation. Moreover, treatment of NSCLC cells with low Src kinase activity with an IGF-1R TKI enhances the reciprocal Src and IGF-1R activation via stabilization of IGF-1R and Src proteins. Finally, we show that Src antagonism universally sensitizes NSCLC cells to IGF-1R TKIs in vitro and in vivo. These results suggest that a combined treatment with IGF-1R and Src inhibitors is a useful and clinically applicable therapeutic strategy for NSCLC.
Co-activation of IGF-1R and Src in human NSCLC
Mutual phosphorylation of IGF-1R and Src in NSCLC cells
Previous reports suggested that Src can directly phosphorylate IGF-1R at the sites of ligand-induced autophosphorylation [12, 13]. Consistent with this finding, in vitro kinase assays showed the ability of Src, derived from A549 cells or recombinant protein (rSrc), to phosphorylate recombinant IGF-1R protein (GST-IGF-1R) (Fig. 2f). Moreover, the Src immunoprecipitates from H226B cells transfected with wild-type Src showed greater IGF-1R phosphorylation than those from the kinase-dead Src (Y416F)-transfected cells (Fig. 2g). These findings indicated that Src can directly phosphorylate IGF-1R, but indirect mechanisms (as a consequence of an autocrine mechanism or the activation of another kinase) may be also involved in Src-induced IGF-1R phosphorylation.
We next assessed the potential involvement of IGF-1R in Src phosphorylation. To this end, we constructed a mutant IGF-1R that replaced tyrosine 1135 with phenylalanine (Y1135F). In contrast to the wild-type receptor, this mutant was unresponsive to IGF-stimulated IGF-1R tyrosine phosphorylation , confirming the importance of the site for receptor activity. Transfection with wild-type IGF-1R but not a mutant IGF-1R (Y1135F) (Fig. 2h) or stimulation with IGF-1 (Fig. 2i) or 10 % FBS (Fig. 2j, left) induced Src phosphorylation (Additional file 6: Figure S5C–S5E). The FBS-induced Src phosphorylation was effectively attenuated by transfection with a shRNA against IGF-1R (Fig. 2j, right; Additional file 6: Figure S5E). An in vitro kinase assay showed that IGF-1R immunoprecipitated from A549 cells phosphorylated Src (Fig. 2k; Additional file 6: Figure S5F). These findings revealed the ability of IGF-1R to phosphorylate Src. Collectively, these results indicated the mutual phosphorylation of IGF-1R and Src in NSCLC cells.
Src-dependent activation of IGF-1R downstream signaling effectors in high-pSrc-expressing NSCLC cells after treatment with IGF-1R TKIs
Linsitinib treatment-induced stabilization of IGF-1R and Src protein leads to the reciprocal co-activation of IGF-1R and Src in low-pSrc-expressing NSCLC cells
Based on the mutual activation of IGF-1R and Src and the Src-dependent activation of Akt in high pSrc-expressing cells after linsitinib treatment, we speculated that cells with a relatively low level of active Src may display high levels of linsitinib sensitivity. However, when we compared the linsitinib sensitivity of a subset of NSCLC cell lines with high or low levels of pSrc expression, we observed a statistically significant inverse correlation between pSrc levels and linsitinib sensitivity (Additional file 11: Figure S10). We then monitored the effects of linsitinib on IGF-1R and Src phosphorylation in low-pSrc-expressing H226B and H460 cells during 5 days of daily treatment with linsitinib (1 μM). We found transient decreases in pIGF-1R and pSrc levels followed by time-dependent increases in pIGF-1R, pSrc, pSTAT3, and pAkt in the linsitinib-treated H226B cells (Fig. 3c; Additional file 9: Figure S8B). In vitro kinase assays showed that IGF-1R and Src in the linsitinib-treated cells had increased capacity for mutual phosphorylation (Fig. 3d). Moreover, combined treatment with linsitinib and dasatinib effectively suppressed the linsitinib-induced activation of IGF-1R, Src, and Akt (Fig. 3e). The linsitinib treatment of H460 and Calu-1 cells also showed an initial decrease followed by a time-dependent increases in IGF-1R, Src, and Akt phosphorylation (Fig. 3c; Additional file 12: Figure S11), which was blocked by the dasatinib treatment (Fig. 3e; Additional file 12: Figure S11). Combined treatment with another SFK inhibitor PP2  also decreased linsitinib-induced IGF-1R, Src, and Akt phoshsphorylation in H460 cells (Additional file 13: Figure S12). These findings suggest that linsitinib treatment augmented the reciprocal co-activation of IGF-1R and Src in low-pSrc-expressing NSCLC cells. Notably, IGF-1R and Src protein expressions were markedly increased in H226B and H460 cells after 3 days of treatment with linsitinib (Fig. 3c). Therefore, we assessed whether the increased levels of IGF-1R and Src proteins in linsitinib-treated cells were due to increased protein synthesis or stability by measuring these protein levels after the linsitinib-treated cells were treated with cycloheximide (CHX). The linsitinib-treated H226B and H460 cells showed a markedly decreased turnover of IGF-1R and Src proteins after 24 h of cycloheximide treatment compared with those treated with vehicle (Fig. 3f). These findings suggested that linsitinib treatment stabilized IGF-1R and Src proteins in low-pSrc-expressing NSCLC cells, ultimately leading to the enhanced reciprocal activation of IGF-1R and Src. Interestingly, in spite of blockade of EGFR phosphorylation, the increases in total and phosphorylated Src and total EGFR expression were found in A549 cells treated with an EGFR TKI erlotinib for 3 days, suggesting that these phenomenon might not be specific to the IGF-1R targeted agents and NSCLC cells may also be able to bypass EGFR by activating Src (Additional file 14: Figure S13).
Src activity mediates de novo linsitinib resistance in NSCLC cells
In accordance with our above findings from the high pSrc-expressing NSCLC cells, linsitinib treatment decreased the viability of low pSrc-expressing NSCLC cells more effectively under low-serum conditions than high-serum conditions (Additional file 15: Figure S14A and S14B). Similar results were observed after treatment with another IGF-1R TKI, AG1024 (Additional file 17: Figure S16). In addition, combined treatment with linsitinib and dasatinib suppressed cell proliferation (Fig. 4f) and anchorage-dependent (Fig. 4g) and anchorage-independent colony formation (Fig. 4h) and increased caspase-3 cleavage (Fig. 4i) in low pSrc-expressing H226B and H460 cells more potently than either drug alone.
Enhanced effects of co-targeting IGF-1R and Src on viability (Additional file 18: Figure S17A), anchorage-dependent (Additional file 18: Figure S17B) and anchorage-independent colony formation (Additional file 18: Figure S17C) were also observed in both high-pSrc-expressing and low-pSrc-expressing NSCLC cells when another pharmacologic SFK inhibitor, PP2 , was combined with linsitinib. Notably, co-targeting Src and IGF-1R effectively suppressed the colony formation of EGFR TKI-resistant NSCLC cells (H1975, PC-9/GR and PC-9/ER)  (Additional file 18: Figure S17D). These results suggest that Src antagonism may broadly increase linsitinib sensitivity in NSCLC cells regardless of IGF-1R and Src activity as well as EGFR TKI resistance.
Targeting Src overcomes IGF-1R TKI resistance in vivo
The IGF-1R pathway plays a major role in mediating the activation of prosurvival pathways and contributes to resistance to various anticancer therapies, including chemotherapies, radiation therapy, and molecular targeted therapies, in multiple cancer types [5–7, 11, 32]. These findings indicate that IGF-1R-targeted therapies represent a promising single-agent or combination regimen. However, NSCLC cells appear to exhibit resistance to the IGF-1R-targeted agents through various mechanisms. With respect to IGF-1R mAb resistance, our laboratory has shown that the inactivation of IGF-1R by the IGF-1R mAb resulted in enhanced signaling through IGF-dependent integrin/Src signaling and the Akt/mTOR-mediated protein synthesis of EGFR and Akt, leading to drug resistance [33, 34]. IR has also been suggested to mediate intrinsic and/or acquired resistance to IGF-1R mAbs [30, 35]. In this regard, dual IGF-1R/IR TKI regimens may be more effective in blocking the IGF-1R pathway in patients with NSCLC. However, the dual IGF-1R/IR TKI linsitinib has shown moderate efficacy and/or severe toxicity in multiple preclinical and clinical studies for various types of cancer3 [36, 37]. Furthermore, the major components of the IGF-1R pathway, such as pIGF-1R, pAkt, pERK, pIRS, and pS6K, could not predict response to linsitinib . Therefore, identifying and targeting the mechanism of resistance against the IGF-1R TKIs would be effective and clinically practical in controlling prosurvival pathways.
De novo and acquired resistance to inhibitors of RTK have been suggested to occur due to the mutation, amplification, and increased/decreased expression of signaling molecules involved in multiple counter-regulatory pathways . However, unlike EGFR, neither activating mutations nor gene amplifications of IGF-1R are common . We have demonstrated that the activation of the IGF-1R pathway through the increased expression of tissue-derived IGFs and the loss of IGFBP-3 is an early event in lung carcinogenesis [18, 41]. However, an analysis of previously studied tissue specimens from NSCLC patients and a panel of NSCLC cell lines revealed no correlation between the expression levels of pIGF-1R and IGFs. The previous reports, including; 1) the mutation and/or overexpression of various molecules involved in Src activation, such as EGFR, KRas, and HER2 [16, 29], has been implicated in the mechanism of resistance to IGF-1R TKIs ; and 2) the aberrant overexpression and activation of IGF-1R and Src in many NSCLCs , and our current results, indicating; 1) a strong positive correlation between pSrc and pIGF-1R in a large TMA of NSCLC tissues and in the majority of NSCLC cell lines; 2) the suppression of EGFR- or integrinβ3 -mediated activation of IGF-1R via dasatinib treatment; and 3) mutual direct phosphorylation between Src and IGF-1R, suggested that Src acts as a key node that link signaling from the cross-talk of several membrane-associated receptors to the ligand-independent activation of IGF-1R in NSCLC.
We then hypothesized that Src plays a critical role in resistance to IGF-1R TKIs in NSCLC cells. In support of the notion, combined treatment with linsitinib and dasatinib was more efficacious in suppressing the proliferation and colony-forming ability of high-pSrc-expressing NSCLC cells than single drug treatments. We further expected that low-pSrc-expressing cells would be more sensitive to IGF-1R TKIs. However, low-pSrc-expressing NSCLC cells showed significantly weaker sensitivity to IGF-1R TKIs than high-pSrc-expressing NSCLC cells. Our subsequent studies provide evidence that the treatment of low-pSrc-expressing cells with an IGF-1R TKI reprograms the cells to stabilize IGF-1R and Src proteins, leading to the enhanced reciprocal coactivation of IGF-1R and Src. Moreover, combined treatment with linsitinib and dasatinib resulted in significantly enhanced antiproliferative effects in these NSCLC cell lines. Importantly, co-targeting IGF-1R and Src exhibited apoptotic activities in vitro in both high-pSrc-expressing and low-pSrc-expressing NSCLC cell lines as well as in vivo in immunodeficient mice bearing xenograft tumors of these cell lines and in those bearing heterotransplant xenogrft tumors from a patient with NSCLC. Collectively, these findings suggest a potential benefit of targeting Src in preventing NSCLC cell resistance to IGF-1R TKIs regardless of IGF-1R or Src activity.
Notably, the combination of linsitinib and dasatinib induces apoptosis in H1975, PC-9R, and PC-9/ER cells with acquired resistance to EGFR TKIs in vitro and causes substantial tumor shrinkage in mice harboring H1975 xenograft tumors in vivo. EGFR-mutant tumors are known to have an increased sensitivity to the first-generation EGFR TKIs gefitinib and erlotinib. However, a secondary T790M mutation promotes the acquisition of resistance to EGFR TKIs in EGFR-mutant tumors. Recent studies suggest that second- and third-generation EGFR inhibitors are more potent than gefitinib or erlotinib (at least in pre-clinical models) against the drug-resistant mutation . In addition, several combination strategies, including the combination of EGFR TKIs with inhibitors of downstream EGFR targets, such as PI3K or STAT5, have been tested in preclinical models of EGFR-mutant NSCLCs . Although these strategies are promising, their clinical efficacy remains to be evaluated. Hence, there is a need for alternative strategies to address the problem of resistance to EGFR TKIs. In this regard, our current findings suggest that the co-inhibition of IGF-1R and Src may provide a substantial therapeutic advantage for patients with NSCLC with acquired resistance to EGFR TKIs as well.
We have demonstrated a potential biological interaction between IGF-1R and Src signalings in NSCLC. We also show that this interaction interferes with the therapeutic activities of IGF-1R TKIs and that the addition of a Src inhibitor is effective in overcoming resistance to IGF-1R TKIs. Because the clinical development of TKIs of IGF-1R and Src is ongoing [45, 46], our findings directly impact the current clinical management of patients with NSCLC. It is noteworthy that a phase I trial of solid tumors treated with XL228 (a small molecule inhibitor targeting Src family kinases, IGF-1R, Aurora A, and Bcr-Abl in the low nanomolar range ) has shown promising preliminary clinical results in melanoma and solid tumors including NSLCLC . Considering the extensive toxicities of Src inhibitors and IGF-1R TKIs [49, 50], further studies are warranted to evaluate the efficacy of co-targeting IGF-1R and Src in additional preclinical and clinical settings.
Human NSCLC cells (A549, H1975, H292, H522, HCC827, Calu-1, H520, H1299, H460, H1993, H2122, and H1703) were purchased from ATCC (Manassas, VA, USA). Other NSCLC cells were kindly provided by Dr. John V. Heymach (MD Anderson Cancer Center, Houston, TX, USA). PC-9R cells (gefitinib-resistant PC-9 cells) were kindly provided by Dr. Mien-Chie Hung (MD Anderson Cancer Center, Houston, TX, USA). PC-9/ER (H) cells (erlotinib-resistant PC-9 cells) were kindly provided by Dr. Jae Cheol Lee (Asan Medical Center, Seoul, Republic of Korea).The cells were maintained in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and antibiotics. The cells were incubated at 37 °C with 5 % CO2 in a humidified atmosphere.
Linsitinib (OSI-906) was provided by OSI Pharmaceuticals, LLC (Melville, New York, USA) or purchased from Selleckchem (Houston, TX, USA). PP2 was purchased from EMD Chemicals (Gibbstown, NJ, USA). Dasatinib was purchased from LC laboratories (Woburn, MA, USA) or the pharmacy in the MD Anderson Cancer Center. Antibodies against pIGF-1R/IR (Y1131), pIGF-1R/IR (Y1135/36), IGF-1R, IR, pEGFR (Y1068), pEGFR (Y845), Src, pSrc (Y416), Met, pAkt, Akt, pERK1/2 (p42/44), ERK and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cleaved PARP, pFAK (Y397), FAK, integrin β1, and integrin β3 were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against actin, IGF-1R (C-20), IR, and EGFR (1005) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against pIR/IGFR (Y1162/63) and pFAK (Y576) were purchased from Invitrogen (Carlsbad, CA, USA). Chemicals unless otherwise indicated were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pIRES2-EGFP-integrin β3 expression vector was kindly provided by Dr. Jung Weon Lee (Seoul National University, Seoul, Republic of Korea). The pBabe-Puro EGFR WT plasmid was purchased from Addgene (catalog # 11011, Cambridge, MA, USA) . The mutant Src (Y527F) expression vector was kindly provided by Dr. Faye M. Johnson (MD Anderson Cancer Center, Houston, TX, USA).
Cells were treated with increasing concentrations of linsitinib or SFK inhibitors (dasatinib or PP2), alone or in combination, for 3 days. Drugs were diluted in 1 % or 10 % FBS-containing media. Cell viability was analyzed by the MTT assay. Data were presented as a percentage of the vehicle-treated control group.
Anchorage-dependent growth inhibition
Cells were seeded into 12- or 6-well plates at a density of 80–150 cells/well and were incubated for 24 h. Cells were treated with linsitinib (2 μM) and dasatinib (0.1 μM) or PP2 (2 μM), either alone or in combination, for 12 ~ 14 days. After incubation, cells were fixed with 100 % methanol for 10 mins at room temperature. Cells were further stained with 0.002 % crystal violet solution or hematoxylin for 1 h at room temperature, and then washed with PBS 3 ~ 5 times. Stained colonies were photographed and counted.
Anchorage-independent growth inhibition
Before the experiment, 1 % base agar was made by diluting 4 % sterile low-melting agar solution with 10 % RPMI and pouring 1 ml of the mixture in 12-well plates. H460, H226B, and H226Br cells were harvested and diluted in the medium at a density of 7 × 103 cells/ml (final concentration of 2 × 103 cells/well). Cells were mixed with sterile 1 % agar solution (final concentration of 0.4 %) and immediately poured onto the base agar. After the agar was completely solidified, test samples diluted in 0.5 ml of complete medium were added to the cells. Cells were further incubated for 12 days at 37 °C and 5 % CO2. The colonies were stained with MTT solution (final 200–500 μg/ml). Stained colonies were photographed and counted.
Cell cycle analysis
Cells (3 × 105 cells/dish in 60 mm dishes) were incubated with test samples for 48 h. All adherent or floating cells were collected and washed twice with PBS. Cells were fixed with 100 % methanol overnight. Fixed cells were washed with PBS, and then stained with 50 μg/ml propidium iodide (PI) solution containing 50 μg/ml RNase A for 30 min at room temperature. Fluorescence intensity was analyzed using a FACSCalibur® flow cytometer (BD Biosciences, San Jose, CA, USA). The percentages of the distributions in distinct phases of the cell cycle were determined using the ModFIT LT V2.0 software.
Annexin V/PI double staining
A549, H1975, PC-9R, and PC-9/ER (H) cells were treated with linsitinib and dasatinib alone or in combination for 3 days. Adherent and floating cells were collected and washed with PBS. Cells were diluted with 1X binding buffer at a density of 1 × 105 cells/0.1 ml and then stained with Annexin V or PI using an Annexin V/PI double staining kit (BD Biosciences) according to the manufacturer’s instruction. Stained cells were analyzed by flow cytometry.
Western blot analysis
Cell lysates were prepared with modified RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.25 % Sodium deoxycholate, 1 % Triton X-100, protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA), and phosphatase inhibitor cocktail (Roche)). Crude lysates were centrifuged at 13,000 rpm for 30 min at 4 °C. Supernatants were collected, and protein concentration was determined by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (10–20 μg) of lysates were subjected to 8–10 % SDS-PAGE. Separated proteins were transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with blocking buffer (5 % BSA in PBS containing 0.1 % Tween-20 (PBST)) for 1 h at room temperature and followed by incubation with primary antibodies diluted in 5 % BSA in PBST (1:1000) overnight at 4 °C. Membranes were washed three times with PBST, and incubated with the corresponding secondary antibodies diluted in 3 % non-fat dry milk in PBST (1:5000) for 1–2 h at room temperature. Membranes were washed three times with PBST and were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific).
Total RNA isolation and RT-PCR were performed as described previously  using the following primer sequences: (sense) 5’-TCTTGAAGGTGAAGATGCACACCA-3’ and (antisense) 5’-AGCGAGCTGACTTGGCAGGCTTGA-3’ for IGF1; (sense) 5’-CATCGTTGAGGAGTGCTGTTT-3’ and (antisense) 5’-GTCTTGGGTGGGTAGAGCAAT-3’ for IGF2; and (sense) 5’-ACTACCTCATGAAGATC-3’ and (antisense) 5’-GATCCACATCTG CTGGAA-3’ for actin. PCR was performed under the following conditions: 95 °C for 5 min (initial denaturation), followed by 28 or 35 cycles of 95°C for 30 sec, 55 (for IGF2 and Actin) or 58 °C (for IGF1) for 30 sec, and 72 °C for 30 sec, and a final elongation cycle of 72 °C for 7 min. PCR products were resolved on a 2 % agarose gel containing RedSafe nucleic acid staining solution (Intron Biotechnology, Seongnam-si, Kyunggi-do, Republic of Korea) and visualized using a Gel Doc EZ System (Bio-Rad Laboratories).
Transfection was conducted with lipofectamine 2000 (Invitrogen) according to the manufacture’s recommended procedure.
In vitro kinase assay
A549 cells were lysed in modified RIPA buffer, and immunoprecipitation was performed via incubation with 0.8 μg of anti-c-Src antibody and 20 μl of protein G agarose. After overnight incubation at 4 °C, the immunoprecipitates were washed three times with lysis buffer, washed three times with kinase buffer (20 mM HEPES (pH 7.5) 10 mM MgCl2, 0.5 mM EGTA, 2 mM DTT, 0.5 mM Na3VO4, and 0.5 M NaF), suspended in 30 μl kinase buffer containing ATP (20 μM) and 200 ng of purified GST-IGF-1R (aa959–1367) as a substrate, and incubated for 30 min at 37 °C for in vitro kinase assays. The reaction products were analyzed via Western blotting with a phospho-IGF-1R (Y1131) antibody.
For IGF-1R kinase assays, 200 ng of purified GST-c-Src (aa1-536) was reacted with immunoprecipitates against IGF-1Rβ in a kinase buffer containing ATP (20 μM) at 37 °C for 30 min and then analyzed via Western blotting with a phospho-Src family (Tyr 416) antibody. Src and IGF-1R kinase activity in linsitinib-treated H460 cells were also determined as described above.
Protein degradation assay
To investigate the effects of linsitinib on IGF-1R and Src protein stability, cells maintained in linsitinib for 5 days were treated with cycloheximide (CHX, 50 μg/ml) for the indicated time, and the total IGF-1R and Src levels were analyzed via Western blotting.
In vivo tumor xenograft model
All animal procedures were performed in accordance with a protocol approved by the Seoul National University Institutional Animal Care and Use Committee (approval Nos. SNU-130426-9 and SNU-130820-6). Mice were fed standard mouse chow and water ad libitum and housed in temperature- and humidity-controlled facilities with a 12-hour light/12-hour dark cycle. H1975 and H460 cells (1 × 107 cells) were subcutaneously injected into the left and right flank of each six week-old female athymic mouse on day 0. For the patient-derived tumor xenograft (PDX) experiment, tumors that were passed 3 times in mice were minced into 2-mm3 pieces and subcutaneously inoculated into NOD/SCID mice. When the tumor volume reached approximately 100–150 mm3, the mice were randomly divided into four groups and treated with linsitinib (25 or 50 mg/kg, oral gavage), dasatinib (10 or 25 mg/kg, oral gavage), or a combination of linsitinib and dasatinib six times per week for 3 weeks. Linsitinib was dissolved in a 25 mM tartaric acid solution. Dasatinib powder was dissolved in an 80 mM citric acid solution or a 100 mg dasatinib tablet was suspended in PBS. Tumor growth was determined by measuring the short and long diameters of the tumors with a caliper every 2 days, and body weight was measured to monitor drug toxicity. The tumor volume was calculated using the following formula: tumor volume (mm3) = (short diameter)2 × (long diameter) × 0.5.
Immunohistochemical analyses to detect PCNA, cleaved caspase-3, pIGF-1R, and pSrc expression in the xenograft and PDX tumors were performed as described previously . In brief, formalin-fixed and paraffin-embedded tissue sections (4 μm) were deparaffinized, dehydrated, and treated with methanol containing 3 % hydrogen peroxide. Slides were incubated with anti-PCNA (Abcam, Cambridge, UK), anti-cleaved caspase-3 (Cell signaling), anti-pIGF-1R (Cell signaling), and anti-pSrc (Cell signaling) antibodies overnight at 4 °C, followed by incubation with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 1 hour at room temperature. Signals were detected using Diaminobenzidine Substrate kit (Vector Laboratories). Slides were counterstained with hematoxylin. The detailed procedures for immunohistochemical analysis and scoring of tissue microarray were described in our previous report .
The data are presented as the means ± SD of a representative of at least two independently performed experiments. Statistical analysis was performed with Microsoft Excel software (Microsoft Corp., Redmond, MA, USA) or GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). The quantitative analysis of blots using densitometry was performed with Image J software (National Institute of Health, Bethesda, MD, USA) . The statistical significance of the data obtained from the in vitro experiments was analyzed using two-sided Student’s t-tests. The statistical significance of data obtained from the in vivo experiments was analyzed via one-way or two-way ANOVA. P values of 0.05 or less were considered to be statistically significant.
Comprehensive Cancer Information, National Cancer Institute, http://www.cancer.gov/cancertopics/pdq/treatment/non-small-cell-lung/healthprofessional/page1, Accessed on September 16, 2014
ClinicalTrials.gov identifiers NCT01016860 and NCT01205685
ClinicaTlrials.gov, the U.S. National Institutes of Health, http://clinicaltrials.gov/ct2/results?term=linsitinib&Search=Search, Accessed on October 7, 2014
This work was supported by the National Institutes of Health grant R01 CA100816, a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; Nos. NRF-2011-0017639 and NRF-2011-0030001), and in part by grants from the National Foundation for Cancer Research, the Department of Defense, and the BATTLE program (W81XWH-06-1-0303).
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