Mechanisms of enhanced radiation response following epidermal growth factor receptor signaling inhibition by erlotinib (Tarceva).
Erlotinib (Tarceva) is an orally available HER1 (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor advancing through clinical trials for the treatment of a range of human malignancies. In this study, we examine the capacity of erlotinib to modulate radiation response and investigate specific mechanisms underlying these interactions in human tumor cell lines and xenografts. The impact of erlotinib on cell cycle kinetics was analyzed using flow cytometry, and the impact on apoptosis was evaluated via fluorescein-labeled pan-caspase inhibition and poly(ADP-ribose) polymerase cleavage. Radiation-induced EGFR autophosphorylation and Rad51 expression were examined by Western blot analysis. Radiation survival was analyzed using a clonogenic assay and assessment of in vivo tumor growth was done using a mouse xenograft model system. Microarray studies were carried out using 20 K human cDNA microarray and select genes were validated using quantitative reverse transcription-PCR (RT-PCR). Independently, erlotinib and radiation induce accumulation of tumor cells in G(1) and G(2)-M phase, respectively, with a reduction of cells in S phase. When combined with radiation, erlotinib promotes a further reduction in S-phase fraction. Erlotinib enhances the induction of apoptosis, inhibits EGFR autophosphorylation and Rad51 expression following radiation exposure, and promotes an increase in radiosensitivity. Tumor xenograft studies confirm that systemic administration of erlotinib results in profound tumor growth inhibition when combined with radiation. cDNA microarray analysis assessing genes differentially regulated by erlotinib following radiation exposure identifies a diverse set of genes deriving from several functional classes. Validation is confirmed for several specific genes that may influence radiosensitization by erlotinib including Egr-1, CXCL1, and IL-1beta. These results identify the capacity of erlotinib to enhance radiation response at several levels, including cell cycle arrest, apoptosis induction, accelerated cellular repopulation, and DNA damage repair. Preliminary microarray data suggests additional mechanisms underlying the complex interaction between EGFR signaling and radiation response. These data suggest that the erlotinib/radiation combination represents a strategy worthy of further examination in clinical trials.
Chinnaiyan
Prakash
P
Department of Human Oncology, University of Wisconsin, 600 Highland Avenue, Madison, WI 53792, USA.
Huang
Shyhmin
S
Vallabhaneni
Geetha
G
Armstrong
Eric
E
Varambally
Sooryanarayana
S
Tomlins
Scott A
SA
Chinnaiyan
Arul M
AM
Harari
Paul M
PM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000532:radiotherapy; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000532:radiotherapy; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000528:radiation effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000528:radiation effects*; D000069347:Erlotinib Hydrochloride; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000532:radiotherapy*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000532:radiotherapy*; D011799:Quinazolines / Q000494:pharmacology*; D051135:Rad51 Recombinase; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D023041:Xenograft Model Antitumor Assays
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D011799:Quinazolines; D011838:Radiation-Sensitizing Agents; D000069347:Erlotinib Hydrochloride; D066246:ErbB Receptors; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse