A431CR cells were treated with cetuximab alone, pertuzumab alone, or a combined mix of both drugs at the indicated concentrations, and viable cells were measured (mean +/? SD) after 6 days treatment. plotted relative to untreated controls. ERBB2 expression is usually confirmed by immunoblotting. Physique S3. ERBB2 maintains ERK 1/2 signaling in the presence of cetuximab. A. HCC827 GFP and HCC827 ERBB2 cells were treated with indicated concentrations of cetuximab for 6 hours. Cell extracts were immunoblotted to detect indicated proteins. GFP; green fluorescent protein B. HCC827 cells expressing GFP or BRAFV600E were treated with cetuximab at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. C. Cells from B. were treated with indicated concentrations of cetuximab for 6 hours. Cell extracts were immunoblotted to detect indicated proteins. D. GEO and GEO CR3 cells were treated with increasing concentrations of AZD6244 and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls Physique S4. Lapatinib restores sensitivity to cetuximab. A. HCC827 CR2 and CR4 cells were treated with cetuximab alone, or in combination with 100 nM lapatinib, at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. HCC827 CR2 cells were treated with cetuximab (10 g/ml) alone, lapatinib alone (100 nM) or with both drugs for 6 hours. Cell extracts were immunoblotted to detect indicated proteins. EsculentosideA Physique S5. Heregulin mediates resistance to cetuximab in A431 cells. A. Parental A431 and resistant A431CR cells were lysed, and the whole cell lysates were hybridized to a phospho-RTK array. In the array, each RTK was spotted in duplicate. Hybridization signals at the corners served as controls. B. A431CR cells express greater amounts of heregulin. Cell extracts were immunoblotted to detect indicated proteins. C. A431 cells were exposed to indicated concentrations of heregulin for 6 hours. Cell extracts were immunoblotted to detect indicated proteins. D. Control or heregulin (HRG) siRNAs were transfected into A431CR cells. EsculentosideA Heregulin mRNA was measured by quantitative PCR as explained in Materials and Methods. *, p= 0.0044 (unpaired test). Cell extracts isolated from your cells were immunoblotted to detect indicated proteins. Figure S6. Heregulin mediates resistance to cetuximab in the GEO cells. A. GEO cells were treated with cetuximab at the indicated concentrations in the presence of heregulin at the indicated concentrations (ng/ml). The percentage of viable cells is shown relative to untreated controls. B. GEO cells were treated with cetuximab (10 g/ml) alone, heregulin alone (20 ng/ml) or the combination. Cells were lysed, and the indicated proteins were detected by MTG8 immunoblotting. Physique S7. Progression free survival for all those CRC patients treated with cetuximab based therapy. Progression free survival for all those (left) CRC patients with (n = 13) and without amplification (n = 220) treated with cetuximab based therapy. (Right) Data for wild type only patients (amplified; n = 11; non-amplified; n = 171). Comparison based on log-rank test. Figure S8. Increased copy number is usually associated with acquired cetuximab resistance. A. Increased copy number in cetuximab resistant tumor specimen. FISH from a baseline tumor specimen (left) and following acquired cetuximab resistance (right). ERBB2 (reddish) and CEP 17 (green). B. Serum levels of the ERBB2 extracellular domain name (ECD) from colorectal malignancy patients before and after cetuximab-based therapy. Dotted collection, 15 ng/ml (cutoff for abnormal). S, single agent cetuximab; C, combination with chemotherapy; PR, partial clinical response; SD, stable disease. Table S1. Characteristics of colorectal malignancy patients used to evaluate impact of amplification. 5FU; 5-Fluorouracil. The cohort consisted of 262 patients; FISH for amplification was possible in 233/262 (89%) of patients. Table S2. Clinical and treatment information on colorectal malignancy patients utilized for plasma ERBB2 extracellular domain name measurements. Table S3. Characteristics of colorectal malignancy patients utilized for plasma and tumor based studies of heregulin. NIHMS337522-product-01.pdf (1.5M) GUID:?46D02B28-12A8-477D-A2E2-F360507F8034 Abstract The epidermal growth factor receptor directed antibody, cetuximab, is an effective clinical therapy for patients with colorectal, head and neck EsculentosideA and non-small cell lung malignancy patients particularly for those with and wild type cancers. Treatment in all patients is limited eventually by the development of acquired resistance but little is known about the underlying mechanism. Here we show, that activation of ERBB2 signaling, either through amplification or through heregulin upregulation, prospects to prolonged ERK 1/2 signaling and.
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