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Experimental Therapeutics |
Departments of Radiation Oncology [A. C., J. S. L.] and Molecular Oncology [N. J. D.], Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts 02129
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), EGFR undergoes dimerization, which activates the intrinsic protein tyrosine kinase via autophosphorylation within its cytoplasmic domain. The tyrosine-autophosphorylated region serves as a binding site for cytoplasmic messenger proteins, which, in turn, leads to downstream activation of major signaling pathways, including the MAPK and PI3-K pathways (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
. In tumors, EGFR has been shown to mediate antiapoptotic and proinvasive effects through activation of these pathways (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
. It is not surprising, then, that there has been considerable interest in antagonizing EGFR-mediated signaling for antitumor effect, both alone and in combination with cytotoxic agents (e.g., radiation and chemotherapy). Available agents include monoclonal antibodies, small molecule tyrosine kinase inhibitors, antisense oligonucleotides, and others (3) . Data from preclinical studies have been encouraging, with evidence that inhibition of EGFR signaling results in enhanced apoptosis, reduced cellular motility/invasive potential, and reduced angiogenic capability (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) . It remains unclear whether all EGFR-expressing tumors of a given histological subtype are equally sensitive to the antitumor effects of EGFR blockade. Indeed, it is unclear whether absolute EGFR expression levels are predictive of the antitumor effects of EGFR blockade.
Malignant gliomas are among the most aggressive and treatment-refractory tumors encountered in the clinic and are known to frequently express/overexpress EGFR (33, 34, 35) . Initial reports suggest that for established, immortalized EGFR-expressing cell lines, blockade of EGFR results in diminution of the malignant phenotype (17 , 19, 20, 21, 22, 23, 24, 25 , 27 , 28 , 30 , 32 , 36) . Whether all EGFR-expressing gliomas are sensitive to the effects of anti-EGFR agents is unclear and has been difficult to study in vitro attributable to the relatively limited number of available EGFR-expressing glioma cell lines. To complicate matters, these established glioma cell lines may differ considerably from the primary tumors from which they were derived. To address this issue, we established in vitro cultures of primary EGFR-expressing glioblastomas. Two of these cell lines had equivalent levels of EGFR expression, yet demonstrated very different sensitivities to inhibition of EGFR signaling despite objective evidence of anti-EGFR effect at the level of the receptor in both cases.
We report here that the IGFR-I can compensate for loss of EGFR function in primary glioma cell lines. We demonstrate here that such a compensation can function to override the expected benefits from loss of EGFR function in gliomas and serves to: (a) inhibit both spontaneous and radiation-induced apoptosis; and (b) enhance invasive capabilities of primary glioblastoma cell lines. These results reveal one mechanism by which certain EGFR-expressing gliomas may demonstrate resistance to the effects of anti-EGFR targeting alone.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western Blot Analysis.
Lysates were generated by placing these cells in RIPA lysis buffer. Bradford assays were performed to determine total protein concentrations, which were normalized to 1 µg/µl for all samples. Samples were then prepared in sample buffer and heated to 95°C for 5 min. The samples were run on 12% polyacrylamide gels for total MAPK (p44/p42) and phospho-MAPK (p44/p42) and on 10% gels for total Akt, phospho-Akt, p70s6k, and phospho p70s6k. Immunoprecipitation/Western analysis for phospho-IGFR-I was performed as described previously (38)
. Protein lysates (15 µl) in sample buffer from each tissue were loaded within each well. Gels were run at constant current (40 mA) for 3–4 h for maximum separation. Wet transfer was performed for 4 h at constant voltage (40 V) using polyvinylidene difluoride membrane presoaked in methanol. The membrane was then blocked for 1 h in 5% milk in 0.2% TBST. The membranes were then washed in 0.2% TBST x 3 for 15 min each. The membranes were then incubated overnight with primary antibodies directed against either phospho-EGFR; total MAPK (p44/p42); phospho-MAPK (p44/p42); total Akt; phospho-Akt; Akt 1, 2, or 3 isoforms; p70s6k; or phospho-p70s6k. Subsequently, the membranes were washed in 0.2% TBST x 3 for 15 min each. The membrane was then incubated with secondary antibody (antirabbit) for 45 min. Chemiluminescent (Bio-Rad) detection was then used to detect phospho-MAPK (p44/p42) and phospho-Akt expression. The expression levels of both were quantitated using densitometry. The primary antibodies directed against phospho-EGFR, total MAPK (p44/p42), phospho-MAPK (p44/p42), total Akt, phospho-Akt, p70s6k, and phospho p70s6k were purchased from Cell Signaling Technology. Primary antibodies directed against Akt 1, 2, and 3 isoforms were purchased from Santa Cruz Biotechnology.
Apoptosis Assay.
The cells were examined for their ability to undergo both spontaneous apoptosis and apoptosis in response to radiation +/- selective pharmacological inhibitors, including: AG1478 (selective EGFR tyrosine kinase inhibitor), AG1024 (selective IGFR-I inhibitor), PD98059 (selective MEK1 inhibitor), LY294002 (selective PI3-K inhibitor), rapamycin (p70s6k inhibitor), and antisense Akt1(ISISR). For the radiation response assay, the cells were irradiated to a single dose of 8 Gy +/- above inhibitors, then apoptosis was assessed at various time points from 2 to 24 h after treatment to assess peak apoptotic index. Controls in each case corresponded to untreated cells. Apoptosis was assayed by detection of membrane externalization of phosphatidylserine with Annexin V-FITC conjugate (Caltag). To assess peak apoptotic index, cells were harvested at various intervals after treatment and resuspended in PBS solution. Both adherent and floating cells were harvested for the apoptosis assay. The cells were then suspended in 1–2 ml of FITC-Annexin V solution. Propidium iodide was added to a final concentration of 1 µg/ml. This was analyzed by flow cytometry using blue light excitation, and green fluorescence of FITC was measured at 530 +/- 20, and red fluorescence was measured at >600.
Invasion Chamber Assay.
Invasion assays were performed using the MatrigelR (Becton Dickinson) basement membrane chamber assay. Before use, the inserts were rehydrated for 1.5–2 h with 0.5 ml of warm DMEM containing 0.1% BSA. Rehydrated chambers (containing MatrigelR Matrix) were placed into the wells of a 24-well companion plate. Each well contained 0.5 ml of media with 5 x 103 cells plated. The chambers were incubated for 24 h at 37°C in a 5% CO2 atmosphere. After 48 h, the chambers were scrubbed with a cotton swab to remove the matrix. Cells on the undersurface of the membrane were fixed and stained in solution containing methylene blue dissolved in 70% ethanol and counted on examination by light microscopy. Control chambers included untreated cells. The remaining chambers contained cells treated by specific pharmacological inhibitors. The percentage of invasive cells was calculated by comparing the number of invasive cells after pharmacological inhibition divided by the number of invasive cells in the control chambers (untreated cells).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, on administration of the potent EGFR tyrosine kinase inhibitor, AG1478, these two cell lines demonstrated significant differences in both spontaneous and radiation-induced apoptosis, as well as a change in invasive potential (Fig. 2, A–E)
. In the GBMS cell line, there was significant enhancement of both spontaneous and treatment-induced apoptosis after AG1478 administration. In contrast, in the GBMR cell line, there was no apparent enhancement of either spontaneous or radiation-induced apoptosis after AG1478 administration. The percentage of invasive cells likewise decreased more significantly in the GBMS versus the GBMR cell line after AG1478. To verify activity of AG1478 against the EGFR tyrosine kinase, levels of phospho-EGFR were measured after AG1478 treatment. It was evident that in both cell lines, the decrease in phospho-EGFR levels after AG1478 was equivalent (Fig. 1A)
. Therefore, the differences in antitumor effect on EGFR blockade in these two GBM cell lines did not appear to stem from lack of activity of AG1478 on the EGFR tyrosine kinase.
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. This appears to suggest that resistance to the effects of EGFR blockade was not mediated through MAPK (p44/p42) signaling for the GBMR cells. Next, we examined activity along the PI3-K pathway (via phospho-Akt levels) in the two cell lines after AG1478 administration. Interestingly, in the GBMS cell line, there was a substantial decrease in phospho-Akt levels on AG1478 administration, whereas in the GBMR cell line, no such decrease was evident (Fig. 1C)
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IGFR-I Can Compensate for Loss of EGFR Signaling in GBM Cell Lines.
We explored the underlying reasons for continued PI3-K signaling in the GBMR cell line after AG1478. PTEN, a suspected down-regulator of PI3-K activation of Akt, demonstrated normal expression levels in the GBMR cell line, making its absence an unlikely cause. Next, we investigated the expression patterns of a panel of known RTKs expressed in GBM tumors that signal through PI3-K: IGFR-I, PDGFR, TrkA, and other Erb-family members. In the GBMS cell line, only expression of Erb-1 (EGFR) was found among these. However, in the GBMR cell line, coexpression of both EGFR and IGFR-I was found (Figs. 1A
and 3A
). Treatment of this cell line with AG1478 resulted in increased levels of both IGFR-I and phospho-IGFR-I (Fig. 3, A and B)
, suggesting a possible compensatory response for loss of EGFR function. To test this hypothesis, we used dual targeting of EGFR and IGFR-I to determine whether we could reduce the malignant phenotype of the GBMR cell lines. Indeed, dual targeting increased both spontaneous and radiation-induced apoptosis (Fig. 4, A and B)
. Furthermore, the invasive potential of these cells decreased substantially. In fact, when IGFR-I was inhibited by AG1024 without coinhibition of EGFR, there was still a significant enhancement of apoptosis and reduction of invasive potential evident, unlike that with EGFR inhibition alone (Fig. 4, C and D)
. This suggests that when coexpressed with EGFR, the IGFR-I pathway may provide dominant prosurvival and proinvasive signaling in GBMR. As would be expected, AG1024 failed to demonstrate any effect on the GBMS cell line, which does not express IGFR-I (Fig. 4, E–G)
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. Apoptosis and invasion assays were then performed. It was evident that exogenous IGF-I was a more potent suppressor of spontaneous, as well as radiation-induced, apoptosis than exogenous EGF in the GBMR cell line (Fig. 5, A and B)
. Furthermore, exogenous IGF-I stimulation enhanced the invasive potential of GBMR cells to a greater extent than did EGF (Fig. 5C)
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compared with 4, A and B
, respectively). Additionally, combined AG1024 and AG1478 reduced phospho-Akt levels in the GBMR cell line greater than that seen with AG1478 alone (data not shown) for the GBMR cell line. In contrast, the selective MEK1 inhibitor, PD98059, +/- AG1478 failed to enhance apoptosis as significantly. Reduction of invasive potential was also more pronounced with LY294002 than with PD98059 administration. This suggests, at least causally, that in the GBMR cell line, IGFR-I-mediated compensation for loss of EGFR function is mediated in a large measure through the PI3-K pathway. To more directly evaluate causality, the GBMR cell line was cultured in serum-free media with exogenous IGF-I +/- LY294002/PD98059. LY294002, unlike PD98059, was clearly able to abrogate the antiapoptotic and proinvasive effects of exogenous IGF-I (Fig. 6, A–C)
. This confirms in a more direct manner that IGFR-I-mediated compensation of EGFR blockade (in this case, absence of ligands that bind EGFR) is dependent more on PI3-K than MAPK signaling.
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demonstrates reduced Akt1 levels in GBMR cells on administration of antisense Akt1 but not with the scrambled sequence. Both antisense Akt1 and rapamycin resulted in a reduction of the antiapoptotic effects of exogenous IGF-I to a similar degree. Both antisense Akt-1 and rapamycin suppressed IGF-I-mediated cellular invasion. However, in each case, inhibition of PI3-K by LY294002 resulted in greater enhancement of apoptosis, especially radiation-induced apoptosis, and reduction of invasive potential than combined inhibition of Akt1 and p70S6K, suggesting that IGFR-I-mediated activation of PI3-K has other contributing downstream mediators.
Akt1 and p70s6k also appear to be critical for prosurvival and proinvasive signaling in GBMS cell line, which expresses EGFR but lacks IGFR-I expression. Increasing concentrations of exogenous EGF reduced both spontaneous and treatment-induced apoptosis (Fig. 7, A and B)
. However, in the presence of exogenous EGF, antisense Akt1 and rapamycin both were able to: (a) partially counter these effects and release GBMS cells from EGF-mediated inhibition of both spontaneous and radiation-induced apoptosis; and (b) reduce invasive capability of these cells (Fig. 7C)
. However, the additive proapoptotic effects, especially after radiation, were significantly less with combined Akt1 and p70s6k antagonism than with PI3-K inhibition. This suggests that EGF-, like IGF-I-, mediated activation of PI3-K may generate additional antiapoptotic signals that are not exclusively through Akt1 and p70s6k.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25% of all GBMs express a mutant form of EGFR, termed the vIII mutant, which lacks the extracellular binding domain and is constitutively active (34)
. Preliminary data suggest that established glioma cell lines (e.g., U87) are sensitive to the effects of EGFR blockade and are more prone to undergo apoptosis in response to radiation (36)
. The issue of whether all EGFR-expressing gliomas would be equally sensitive to the effects of EGFR antagonism regardless of expression levels has been difficult to address with the relatively limited number of available EGFR-expressing established glioma cell lines and, therefore, has been poorly understood. Clinical data on squamous cell carcinomas of the head and neck suggest that there may, indeed, be heterogeneity in response to EGFR antagonism among EGFR-expressing tumors (19) . We find that, despite having equivalent EGFR expression levels, primary glioma cell lines can have very different sensitivities to EGFR antagonism. The results described here suggest that signaling through the IGFR-I represents at least one mechanism by which tumor cells can become resistant to anti-EGFR therapy.
It is known that on binding to its substrate, IGF-I, IGFR-I activates the intrinsic tyrosine kinase, resulting in receptor autophosphorylation and the presentation of suitable binding sites for substrates containing either Src homology 2 or phosphotyrosine binding domains (39) . Activation of the SHC/Grb2/SOS/Ras/raf/MEK/MAPK and IRS/PI3-K/Akt/p70s6k pathways is a known consequence of IGFR-I-mediated signal transduction, leading to a host of effects, including: proliferation, tumorigenesis, and inhibition of apoptosis. It appears that in the GBMR cell line, IGFR-I-mediated PI3-K activation results in potent antiapoptotic and proinvasive effects.
Several studies have shown previously that the PI3-K pathway is critical in mediating neuronal cell survival, as well as the survival of other specific tumor subtypes (40, 41, 42, 43) . The downstream mediators of PI3-K function are not entirely clear but are becoming better understood. It is thought that the protein-serine/threonine kinases, 3'-phosphoinositide-dependent kinase-1 and Akt/protein kinase B, are two important mediators of PI3-K signaling (44, 45, 46, 47, 48, 49) .
Akt promotes cell survival in a number of different ways. It phosphorylates the proapoptotic protein BAD so that it is unable to bind and inactivate the antiapoptotic protein, Bcl-xl. Akt also serves to inhibit apoptosis by inhibiting caspase 9 and FKHLR1 (forkhead transcription factor) and by activating IkB kinase (45 , 49, 50, 51) . In addition, Akt has been shown to be the key mediator by which growth factor-growth factor receptor interactions affect the phosphorylation state of mTOR. It has been shown that mTOR has at least two important targets, including 4E-BP1/PHAS-1, which inhibits initiation of translation through its association with eIF-4F (26) . The second target of mTOR is the kinase p70s6k (45) . In a recent study, it was demonstrated that p70s6k may, in fact, be a more potent kinase for BAD than Akt in response to IGF-I stimulation and, hence, may play an important antiapoptotic role (50) .
Given that p70s6k can be phosphorylated by Akt-dependent and independent mechanisms (through phosphoinositide-dependent kinase-1), we proceeded to examine the relative importance of Akt-dependent p70s6k signaling. We found that inhibition of the Akt-dependent pathway by rapamycin antagonizes the antiapoptotic, proinvasive effect of exogenous IGF-I. Although antisense Akt1 also appears to increase apoptosis in both normal astrocytes and tumor cells, antagonism of p70s6k gives this effect almost exclusively in tumor cells (data not shown), suggesting that targeting downstream effectors of Akt, such as p70s6k, may provide a greater therapeutic gain in cases where continued signaling through PI3-K generates resistance to anti-EGFR therapy.
IGFR-I-mediated activation of Akt and p70s6k also appears to play an important role in resistance to radiation-induced apoptosis in primary glioma cell lines. Additionally intriguing is the observation that direct inhibition of PI3-K signaling mediated by IGF-I and EGF (by LY294002) appears to generate a significantly stronger proapoptotic effect after radiation than combined antagonism of Akt1 and p70s6k, which suggests that PI3-K may induce other, heretofore unidentified, downstream prosurvival pathways that are Akt independent.
Our data suggest that simple measurement of absolute EGFR expression levels may be inadequate by itself in predicting which tumors will respond most favorably to anti-EGFR therapy. IGFR-I signaling through PI3-K represents one mechanism of treatment resistance in response to EGFR antagonism. It is possible that other mechanisms of resistance exist, and additional studies are clearly needed to clarify this issue. In specific cases, cotargeting strategies may enhance overall therapeutic effect. As strategies to antagonize EGFR are now becoming reality in the clinic, it is highly desirable to better understand the molecular profiles of tumors that are most likely to respond to such therapeutic strategies and, perhaps proactively, develop strategies to overcome potential mechanisms of resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant K08CA82163, the Massachusetts General Hospital Brian Silber Memorial Fund (both to A. C.), and Grant RO1CA64402 (to N. J. D.), all from the NIH/National Cancer Institute. 
2 To whom requests for reprints should be addressed, at Massachusetts General Hospital, Laboratory of Molecular Oncology, 13th Street, Building 149, Room 7330, Charlestown, MA 02129. Fax: (617) 726-5637; E-mail: achakravarti{at}partners.org
3 The abbreviations used are: EGFR, epidermal growth factor receptor; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; PI3-K, phosphoinositide 3-kinase; IGFR-I, insulin-like growth factor recpetor I; GBM, glioblastoma multiforme; IGF-I, insulin-like growth factor I; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; TBST, Tris buffer solution with 0.2% Triton; mTOR, mammalian target of rapamycin.
Received 8/27/01. Accepted 11/ 1/01.
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F. Morgillo, J. K. Woo, E. S. Kim, W. K. Hong, and H.-Y. Lee Heterodimerization of Insulin-like Growth Factor Receptor/Epidermal Growth Factor Receptor and Induction of Survivin Expression Counteract the Antitumor Action of Erlotinib. Cancer Res., October 15, 2006; 66(20): 10100 - 10111. [Abstract] [Full Text] [PDF]
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S. Massarweh, C. K. Osborne, S. Jiang, A. E. Wakeling, M. Rimawi, S. K. Mohsin, S. Hilsenbeck, and R. Schiff Mechanisms of Tumor Regression and Resistance to Estrogen Deprivation and Fulvestrant in a Model of Estrogen Receptor-Positive, HER-2/neu-Positive Breast Cancer Cancer Res., August 15, 2006; 66(16): 8266 - 8273. [Abstract] [Full Text] [PDF]
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A. Chakravarti, M. G. Erkkinen, U. Nestler, R. Stupp, M. Mehta, K. Aldape, M. R. Gilbert, P. McL. Black, and J. S. Loeffler Temozolomide-Mediated Radiation Enhancement in Glioblastoma: A Report on Underlying Mechanisms Clin. Cancer Res., August 1, 2006; 12(15): 4738 - 4746. [Abstract] [Full Text] [PDF]
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A. A. Adjei Novel combinations based on epidermal growth factor receptor inhibition. Clin. Cancer Res., July 15, 2006; 12(14): 4446s - 4450s. [Abstract] [Full Text] [PDF]
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F. Cappuzzo, L. Toschi, G. Tallini, G. L. Ceresoli, I. Domenichini, S. Bartolini, G. Finocchiaro, E. Magrini, G. Metro, A. Cancellieri, et al. Insulin-like growth factor receptor 1 (IGFR-1) is significantly associated with longer survival in non-small-cell lung cancer patients treated with gefitinib Ann. Onc., July 1, 2006; 17(7): 1120 - 1127. [Abstract] [Full Text] [PDF]
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J. N. Sarkaria, B. L. Carlson, M. A. Schroeder, P. Grogan, P. D. Brown, C. Giannini, K. V. Ballman, G. J. Kitange, A. Guha, A. Pandita, et al. Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin. Cancer Res., April 1, 2006; 12(7 Pt 1): 2264 - 2271. [Abstract] [Full Text] [PDF]
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D. A. Reardon, J. A. Quinn, J. J. Vredenburgh, S. Gururangan, A. H. Friedman, A. Desjardins, S. Sathornsumetee, J. E. Herndon II, J. M. Dowell, R. E. McLendon, et al. Phase 1 Trial of Gefitinib Plus Sirolimus in Adults with Recurrent Malignant Glioma Clin. Cancer Res., February 1, 2006; 12(3): 860 - 868. [Abstract] [Full Text] [PDF]
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V. C. Russo, P. D. Gluckman, E. L. Feldman, and G. A. Werther The Insulin-Like Growth Factor System and Its Pleiotropic Functions in Brain Endocr. Rev., December 1, 2005; 26(7): 916 - 943. [Abstract] [Full Text] [PDF]
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S. J. Isakoff, J. A. Engelman, H. Y. Irie, J. Luo, S. M. Brachmann, R. V. Pearline, L. C. Cantley, and J. S. Brugge Breast Cancer-Associated PIK3CA Mutations Are Oncogenic in Mammary Epithelial Cells Cancer Res., December 1, 2005; 65(23): 10992 - 11000. [Abstract] [Full Text] [PDF]
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J. N. Rich, S. Sathornsumetee, S. T. Keir, M. W. Kieran, A. Laforme, A. Kaipainen, R. E. McLendon, M. W. Graner, B.K. A. Rasheed, L. Wang, et al. ZD6474, a Novel Tyrosine Kinase Inhibitor of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor, Inhibits Tumor Growth of Multiple Nervous System Tumors Clin. Cancer Res., November 15, 2005; 11(22): 8145 - 8157. [Abstract] [Full Text] [PDF]
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S. Thomson, E. Buck, F. Petti, G. Griffin, E. Brown, N. Ramnarine, K. K. Iwata, N. Gibson, and J. D. Haley Epithelial to Mesenchymal Transition Is a Determinant of Sensitivity of Non-Small-Cell Lung Carcinoma Cell Lines and Xenografts to Epidermal Growth Factor Receptor Inhibition Cancer Res., October 15, 2005; 65(20): 9455 - 9462. [Abstract] [Full Text] [PDF]
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S. J. Cohen, R. B. Cohen, and N. J. Meropol Targeting Signal Transduction Pathways in Colorectal Cancer--More Than Skin Deep J. Clin. Oncol., August 10, 2005; 23(23): 5374 - 5385. [Abstract] [Full Text] [PDF]
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Y. Wang, J. Hailey, D. Williams, Y. Wang, P. Lipari, M. Malkowski, X. Wang, L. Xie, G. Li, D. Saha, et al. Inhibition of insulin-like growth factor-I receptor (IGF-IR) signaling and tumor cell growth by a fully human neutralizing anti-IGF-IR antibody Mol. Cancer Ther., August 1, 2005; 4(8): 1214 - 1221. [Abstract] [Full Text] [PDF]
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H E Jones, J M W Gee, K M Taylor, D Barrow, H D Williams, M Rubini, and R I Nicholson Development of strategies for the use of anti-growth factor treatments Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S173 - S182. [Abstract] [Full Text] [PDF]
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N. L. Spector, W. Xia, H. Burris III, H. Hurwitz, E. C. Dees, A. Dowlati, B. O'Neil, B. Overmoyer, P. K. Marcom, K. L. Blackwell, et al. Study of the Biologic Effects of Lapatinib, a Reversible Inhibitor of ErbB1 and ErbB2 Tyrosine Kinases, on Tumor Growth and Survival Pathways in Patients With Advanced Malignancies J. Clin. Oncol., April 10, 2005; 23(11): 2502 - 2512. [Abstract] [Full Text] [PDF]
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J. Baselga and C. L. Arteaga Critical Update and Emerging Trends in Epidermal Growth Factor Receptor Targeting in Cancer J. Clin. Oncol., April 10, 2005; 23(11): 2445 - 2459. [Abstract] [Full Text] [PDF]
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N. J. Meropol Epidermal Growth Factor Receptor Inhibitors in Colorectal Cancer: It's Time to Get Back on Target J. Clin. Oncol., March 20, 2005; 23(9): 1791 - 1793. [Full Text] [PDF]
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C. Tuccillo, M. Romano, T. Troiani, E. Martinelli, F. Morgillo, F. De Vita, R. Bianco, G. Fontanini, R. A. Bianco, G. Tortora, et al. Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor-2 and Epidermal Growth Factor Receptor Small Molecule Tyrosine Kinase Inhibitor, in Combination with SC-236, a Cyclooxygenase-2 Inhibitor Clin. Cancer Res., February 1, 2005; 11(3): 1268 - 1276. [Abstract] [Full Text] [PDF]
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R. K. Goudar, Q. Shi, M. D. Hjelmeland, S. T. Keir, R. E. McLendon, C. J. Wikstrand, E. D. Reese, C. A. Conrad, P. Traxler, H. A. Lane, et al. Combination therapy of inhibitors of epidermal growth factor receptor/vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RAD001) offers improved glioblastoma tumor growth inhibition Mol. Cancer Ther., January 1, 2005; 4(1): 101 - 112. [Abstract] [Full Text] [PDF]
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E. R. Camp, J. Summy, T. W. Bauer, W. Liu, G. E. Gallick, and L. M. Ellis Molecular Mechanisms of Resistance to Therapies Targeting the Epidermal Growth Factor Receptor Clin. Cancer Res., January 1, 2005; 11(1): 397 - 405. [Abstract] [Full Text] [PDF]
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H E Jones, L Goddard, J M W Gee, S Hiscox, M Rubini, D Barrow, J M Knowlden, S Williams, A E Wakeling, and R I Nicholson Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells Endocr. Relat. Cancer, December 1, 2004; 11(4): 793 - 814. [Abstract] [Full Text] [PDF]
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S. Huang, E. A. Armstrong, S. Benavente, P. Chinnaiyan, and P. M. Harari Dual-Agent Molecular Targeting of the Epidermal Growth Factor Receptor (EGFR): Combining Anti-EGFR Antibody with Tyrosine Kinase Inhibitor Cancer Res., August 1, 2004; 64(15): 5355 - 5362. [Abstract] [Full Text] [PDF]
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D. Raben, C. Bianco, V. Damiano, R. Bianco, D. Melisi, C. Mignogna, F. P. D'Armiento, L. Cionini, A. R. Bianco, G. Tortora, et al. Antitumor activity of ZD6126, a novel vascular-targeting agent, is enhanced when combined with ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, and potentiates the effects of radiation in a human non-small cell lung cancer xenograft model Mol. Cancer Ther., August 1, 2004; 3(8): 977 - 983. [Abstract] [Full Text] [PDF]
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L. Castillo, M. C. Etienne-Grimaldi, J. L. Fischel, P. Formento, N. Magne, and G. Milano Pharmacological background of EGFR targeting Ann. Onc., July 1, 2004; 15(7): 1007 - 1012. [Abstract] [Full Text] [PDF]
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A. Chakravarti, G. Zhai, Y. Suzuki, S. Sarkesh, P. M. Black, A. Muzikansky, and J. S. Loeffler The Prognostic Significance of Phosphatidylinositol 3-Kinase Pathway Activation in Human Gliomas J. Clin. Oncol., May 15, 2004; 22(10): 1926 - 1933. [Abstract] [Full Text] [PDF]
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F. Cappuzzo In Reply: J. Clin. Oncol., May 15, 2004; 22(10): 2036 - 2037. [Full Text] [PDF]
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D. Lu, H. Zhang, D. Ludwig, A. Persaud, X. Jimenez, D. Burtrum, P. Balderes, M. Liu, P. Bohlen, L. Witte, et al. Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody J. Biol. Chem., January 23, 2004; 279(4): 2856 - 2865. [Abstract] [Full Text] [PDF]
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F. Ciardiello, R. Bianco, R. Caputo, R. Caputo, V. Damiano, T. Troiani, D. Melisi, F. De Vita, S. De Placido, A. R. Bianco, et al. Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy Clin. Cancer Res., January 15, 2004; 10(2): 784 - 793. [Abstract] [Full Text] [PDF]
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C. L. Arteaga EGF Receptor As a Therapeutic Target: Patient Selection and Mechanisms of Resistance to Receptor-Targeted Drugs J. Clin. Oncol., December 1, 2003; 21(90230): 289s - 291. [Abstract] [Full Text] [PDF]
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A. Weber, C. Huesken, E. Bergmann, W. Kiess, N. M. Christiansen, and H. Christiansen Coexpression of Insulin Receptor-Related Receptor and Insulin-Like Growth Factor 1 Receptor Correlates with Enhanced Apoptosis and Dedifferentiation in Human Neuroblastomas Clin. Cancer Res., November 15, 2003; 9(15): 5683 - 5692. [Abstract] [Full Text] [PDF]
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B. Li, C.-M. Chang, M. Yuan, W. G. McKenna, and H.-K. G. Shu Resistance to Small Molecule Inhibitors of Epidermal Growth Factor Receptor in Malignant Gliomas Cancer Res., November 1, 2003; 63(21): 7443 - 7450. [Abstract] [Full Text] [PDF]
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N. Shinojima, K. Tada, S. Shiraishi, T. Kamiryo, M. Kochi, H. Nakamura, K. Makino, H. Saya, H. Hirano, J.-i. Kuratsu, et al. Prognostic Value of Epidermal Growth Factor Receptor in Patients with Glioblastoma Multiforme Cancer Res., October 15, 2003; 63(20): 6962 - 6970. [Abstract] [Full Text] [PDF]
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 D. E. Hansel, A. Rahman, M. Hidalgo, P. J. Thuluvath, K. D. Lillemoe, R. Shulick, J.-L. Ku, J.-G. Park, K. Miyazaki, R. Ashfaq, et al. Identification of Novel Cellular Targets in Biliary Tract Cancers Using Global Gene Expression Technology Am. J. Pathol., July 1, 2003; 163(1): 217 - 229. [Abstract] [Full Text] [PDF]
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D. H. Johnson and C. L. Arteaga Gefitinib in Recurrent Non-Small-Cell Lung Cancer: An IDEAL Trial? J. Clin. Oncol., June 15, 2003; 21(12): 2227 - 2229. [Full Text] [PDF]
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C. L. Arteaga and J. Baselga Clinical Trial Design and End Points for Epidermal Growth Factor Receptor-targeted Therapies: Implications for Drug Development and Practice Clin. Cancer Res., May 1, 2003; 9(5): 1579 - 1589. [Full Text] [PDF]
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D. Sachdev, S.-L. Li, J. S. Hartell, Y. Fujita-Yamaguchi, J. S. Miller, and D. Yee A Chimeric Humanized Single-Chain Antibody against the Type I Insulin-like Growth Factor (IGF) Receptor Renders Breast Cancer Cells Refractory to the Mitogenic Effects of IGF-I Cancer Res., February 1, 2003; 63(3): 627 - 635. [Abstract] [Full Text] [PDF]
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A. Hurbin, L. Dubrez, J.-L. Coll, and M.-C. Favrot Inhibition of Apoptosis by Amphiregulin via an Insulin-like Growth Factor-1 Receptor-dependent Pathway in Non-small Cell Lung Cancer Cell Lines J. Biol. Chem., December 13, 2002; 277(51): 49127 - 49133. [Abstract] [Full Text] [PDF]
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