Target validation for genomics using peptide‐specific phage antibodies: A study of five gene products overexpressed in colorectal cancer

Abstract

Genomic approaches are providing a wealth of information on differential gene expression in cancer. To identify the most interesting genes amongst the many identified, highthroughput methods for analysis of genes at the translational level are required. We have used a rapid method for the in vitro selection of antibodies to peptide antigens for the generation of probes to 5 gene products that we have found to be overexpressed in colorectal cancer. The rationale of our study was to select a non-immune phage displayed human antibody library on peptides designed from the coding regions of the gene sequences and to verify whether such antibodies would be suitable probes for the parental protein in immunohistochemical and Western blot analysis. After the generation of a profile of genes overexpressed in primary colorectal cancer (CRC) we selected 5 genes, Ese-3b, Fls353, PBEF, SPARC and Smad5 for a more detailed analysis using phage display-derived antibodies. For these 5 antigens we designed 14 -20 amino acid peptides predicted to be exposed on the surface of the parental protein. Selection of a large phage displayed antibody library resulted in specific antibodies for 6 of 8 different peptides with between 2 and 15 different antibodies isolated per peptide. Of 20 antibodies tested, 2 antibodies recognized the putative parental protein from primary CRC tissue. An antibody specific for a PBEF-derived peptide (Fab/PBEF-D4) was shown to recognize a protein product of the expected molecular weight in Western blotting and showed overexpression in n ‫؍‬ 6/8 matched tumor/ normal protein lysates. Furthermore, in immunohistochemistry this antibody showed restricted staining of the tumor stromal compartment with no detectable staining of epithelial cells. The discovery that PBEF is overexpressed in cancer is unexpected given that the normal function of PBEF is as a cytokine required for the maturation of B cell precursors. We also report on the isolation of an antibody (Fab/SMAD-50) specific for a Smad5-derived peptide that showed cytoplasmic staining of epithelial cells in both CRC tumor and matched normal mucosa. Fab/SMAD-50 also bound to a group of proteins in Western blotting with molecular weights consistent with belonging to the Smad family. These antibodies may be suitable probes for further investigation of the roles of PBEF and Smad5 in cancer. The amenability of phage display to automation suggests that this approach may be developed for implementation on a genomics scale. Indeed, the large-scale generation of antibody probes that can be used to study protein expression in situ would be of great value in target validation for functional genomics.

NAMPT overexpression in prostate cancer and its contribution to tumor cell survival and stress response

“…Therefore, more in vivo studies are needed for comprehensive understanding of roles of SIRT1 in cancer. Our finding that SIRT1 is an important downstream effector of NAMPT for oxidative stress in prostate cancer cells is in agreement with the previous finding in normal human vascular smooth muscle cells (van der Veer et al, 2007). However, our study does not exclude the contribution of other sirtuins.…”

“…Increased expression of NAMPT was previously reported in colorectal cancer (Hufton et al, 1999;Van Beijnum et al, 2002). A specific small molecule inhibitor of NAMPT, FK866, inhibits cancer cell growth and induces apoptosis by NAD þ depletion (Hasmann and Schemainda, 2003).…”

“…NAMPT has a crucial role for growth factor-induced myeloid differentiation (Skokowa et al, 2009), circadian clock (Nakahata et al, 2009;Ramsey et al, 2009), glucose-restriction-impaired skeletal myoblast differentiation (Fulco et al, 2008) and insulin secretion in pancreatic beta cells (Revollo et al, 2007). Overexpression of NAMPT extends lifespan of human vascular smooth muscle cells (van der Veer et al, 2007), resembling the effect of yeast NAD þ salvage pathway gene PNC1 for promoting longevity (Anderson et al, 2003).…”

“…SIRT1 promotes mammalian cell survival under oxidative and genotoxic stresses through deacetylation of multiple substrates including p53 (Luo et al, 2001;Vaziri et al, 2001) and forkhead box, class 'O' (FOXO) transcriptional factors (Brunet et al, 2004;Daitoku et al, 2004;Motta et al, 2004). SIRT1 serves as a key physiological downstream effector for NAMPT in regulating cell survival, differentiation and circadian clock (van der Veer et al, 2007;Fulco et al, 2008;Nakahata et al, 2009;Ramsey et al, 2009;Skokowa et al, 2009).…”

Circulating levels of novel adipocytokines in patients with colorectal cancer

“…PBEF1 (PBEF1/Nampt/Visfatin) has been reported to be overexpressed in primary CRC [22,29]. Later, in agreement with our results, Nakajima et al found that visfatin levels in patients with CRC were significantly higher than those of the controls [9].…”