Loss-of-function screening by randomized intracellular antibodies: identification of hnRNP-K as a potential target for metastasis.
We have developed a loss-of-function screening system on the basis of intracellular expression of single domain antibodies. We demonstrate its use in identification of potential targets of metastasis of human cancerous cells. Randomized intracellular antibodies were expressed in highly metastatic cells, and a derivative pool of cells with loss of migration phenotype in chemotaxis assay was isolated. Isolation of antibodies from cells with loss of migration phenotype and identification of their target proteins revealed the involvement of the heterogeneous nuclear ribonucleoprotein K (hnRNP-K), a multifunctional signaling protein, in metastasis. Furthermore, we found that the cytoplasmic accumulation of hnRNP-K is crucial for its role in metastasis. The results demonstrate (i) the advantages of our functional interference screening over the gene-knockouts and gene-silencing, (ii) hnRNP-K as a potential target of metastasis, and (iii) a potential anti-metastasis peptide validated in in vitro cell migration assays.
Inoue
Atsushi
A
National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan. a.inoue@aist.go.jp
Sawata
Shinya Y
SY
Taira
Kazunari
K
Wadhwa
Renu
R
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000276:immunology* / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D002469:Cell Separation; D003593:Cytoplasm / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D012261:Ribonucleoproteins / Q000276:immunology* / Q000378:metabolism*
D000906:Antibodies; D014408:Biomarkers, Tumor; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D012261:Ribonucleoproteins; C072074:HNRNPK protein, human