Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-α, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA-mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC. Clin Cancer Res; 16(13); 3378-89. ©2010 AACR.
The Hedgehog pathway is one of the major driver pathways in pancreatic ductal adenocarcinoma. This study investigated prognostic importance of Hedgehog signaling pathway in pancreatic cancer patients who underwent a radical resection. Tumors and adjacent non-neoplastic pancreatic tissues were obtained from 45 patients with histologically verified pancreatic cancer. The effect of experimental taxane chemotherapy on the expression of Hedgehog pathway was evaluated in vivo using a mouse xenograft model prepared using pancreatic cancer cell line Paca-44. Mice were treated by experimental Stony Brook Taxane SB-T-1216. The transcript profile of 34 Hedgehog pathway genes in patients and xenografts was assessed using quantitative PCR. The Hedgehog pathway was strongly overexpressed in pancreatic tumors and upregulation of SHH, IHH, HHAT and PTCH1 was associated with a trend toward decreased patient survival. No association of Hedgehog pathway expression with KRAS mutation status was found in tumors. Sonic hedgehog ligand was overexpressed, but all other downstream genes were downregulated by SB-T-1216 treatment in vivo. Suppression of HH pathway expression in vivo by taxane-based chemotherapy suggests a new mechanism of action for treatment of this aggressive tumor.
It is anticipated that this project will contribute to a greater understanding of the factors that might empower people in their cancer journey. An empowerment lens, that asks how those affected by cancer may be further enabled at an individual, family/whānau, employer and healthcare professional level may be useful in assisting people navigate and manage their cancer.
Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a poor prognosis, and no targeted therapy is currently available. The aim of the present study was to investigate the prognostic significance of the expression of V-Ki-ras2 Κirsten rat sarcoma viral oncogene homolog (KRAS), downstream signaling pathway genes and the association with clinical characteristics in PDAC patients undergoing radical surgery. Tumors and adjacent non-neoplastic pancreatic tissues were examined in 45 patients with histologically verified PDAC. KRAS and B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene mutation analysis was performed using the KRAS/BRAF/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α array. The transcript profile of 52 KRAS downstream signaling pathway genes was assessed using quantitative-polymerase chain reaction. KRAS mutation was detected in 80% of cases. The genes of four signaling pathways downstream of KRAS, including the phosphoinositide 3-kinase/3-phosphoinositide-dependent protein kinase 1/V-akt murine thymoma viral oncogene homolog 1, RAL guanine nucleotide exchange factor, Ras and Rab interactor 1/ABL proto-oncogene-1, non-receptor tyrosine kinase, and RAF proto-oncogene serine/threonine-protein kinase/mitogen-activated protein kinase pathways, exhibited differential expression in PDAC compared with that in the adjacent normal tissues. However, no significant differences in expression were evident between patients with KRAS-mutated and wild-type tumors. The expression of KRAS downstream signaling pathways genes did not correlate with angioinvasion, perineural invasion, grade or presence of lymph node metastasis. Additionally, the presence of KRAS mutations was not associated with overall survival. Among the KRAS downstream effective signaling pathways molecules investigated, only v-raf-1 murine leukemia viral oncogene homolog 1 expression was predictive of prognosis. Overall, KRAS mutation is present in the majority of cases of PDAC, but is not associated with changes in the expression of KRAS downstream signaling pathways and the clinical outcome. This may partly explain the failure of KRAS-targeted therapies in PDAC.
e14022 Background: Personalised cancer medicine based on mutation profiling of individual tumours is now a reality in the treatment of metastatic colorectal cancer (CRC) since the discovery of mutant KRAS status and resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. However, up to 65% of patients with KRAS wildtype tumours are resistant to anti-EGFR therapy. Mutations in other downstream effectors of the EGFR signaling pathway may also have a negative impact on patient response to anti-EGFR therapy. This study reportsthe analytical performance of a biochip array for the rapid simultaneous detection of 20 mutations within KRAS (including codons 12, 13, 61 and 146), BRAF and PIK3CA genes in CRC tissue samples. Methods: DNA from frozen CRC tissue samples was analysed using the Evidence Investigator KRAS/BRAF/PIK3CA Array. The assay is based on a combination of multiplex PCR and biochip array hybridisation, which enables high multiplexing detection. Innovative PCR priming technology permits high discrimination between multiple wildtype and mutant DNA regions. Providing there are enough copies of DNA present, approximately 1% of mutant can readily be detected in a background of wildtype genomic DNA. Analysis can be completed, from template DNA, through PCR (single reaction) to data readout in less than 3 hours. Confirmation of assay results was performed using Sanger sequencing and pyrosequencing. Results: Mutations were detected in 56% (14/25) of CRC tissue samples assessed. 6 samples were positive for KRAS, 3 for BRAF and 6 for PIK3CA with one sample harbouring both a KRAS and PIK3CA mutation. KRAS targets detected also included much rarer codon 61 and 13 mutations. Sanger sequencing confirmed all positive results apart from two, which were assessed via pyrosequencing. Conclusions: These findings demonstrate the applicability of the KRAS/BRAF/PIK3CA Array to rapidly simultaneously detect mutations within these genes in CRC tissue. The detection of additional markers besides KRAS and extending the profile of this gene to include other codons beyond 12 and 13 may aid in the selection of candidate patients to receive anti-EGFR therapy thereby maximising drug efficacy and minimising adverse patient effects.
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